Haematococcus sp. strains for efficient biomass production using greenhouse gases

ABSTRACT

The invention is directed to Haematococcus sp. KAU-01 as well as to a culture medium for Haematococcus sp. KAU-01, and to methods for using this strain to process environmental pollutants such as gases generated by coal-fired plants.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. application Ser. No. 17/113,816,filed Dec. 7, 2020, now U.S. Pat. No. 11,725,219 entitled BIOFIXATION OFGREENHOUSE GAS BY MASS CULTURE OF HAEMATOCOCCUS SP. KAU-01 MICROALGA INHIGH EFFICIENCY MEDIUM which is hereby incorporated by reference.

BACKGROUND Field of the Invention

The invention pertains to algology and environmental science, especiallyto new microalgae strains that efficiently metabolize carbon dioxide andother greenhouse gases, such as flue gases produced by coal-fired powerplants.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventor(s), to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

DESCRIPTION OF RELATED ART

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventor(s), to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

It is well known that carbon dioxide is an important greenhouse gases(“GHGs”) which can persist in the atmosphere for a long time. Carbondioxide concentrations in air are increasing due to combustion of coal,petroleum, natural gas, and other fuels to produce energy. The increasedatmospheric concentration of carbon dioxide plays a major role in globalclimate change, global warming, ocean acidification and other malignantenvironmental problems.

Average planetary surface temperatures correlate with increases inatmospheric carbon dioxide and can lead to alterations in precipitationpatterns, rises in sea level, and accelerated glacial melting.

Coal-fired power plants release carbon dioxide and other flue andgreenhouse gases into the environment. Biofixation has been proposed asone way to reduce the emissions of gas produced by combustion into theatmosphere. Carbon dioxide and other combustion gases can be used asfeedstocks to culture microorganisms such as microalgae that fixing themin organic materials such as biodiesel fuels, animal feeds, aquaculturefeeds, and other high-valued materials such as pigments andnutraceutical products including astaxanthin; Cheng, J., et al.,Gradient domestication of Haematococcus pluvialis mutant with 15% CO ₂to promote biomass growth and astaxanthin yield, BIORESOUR. TECHNOL.,2016, 216, 340-344; Williams, P. J. et al., Microalgae as biodiesel &biomass feedstocks: review & analysis of the biochemistry, energetics &economics. ENERGY ENVIRON. SCI. 2010, 3: 554-590; and Zheng, Q., et al.,Energy efficient transfer of carbon dioxide from flue gases to microalgasystems. ENERGY ENVIRON. SCI, 2016, 9: 1074-1082. Consequently, it is ofcritical importance to design and formulate a simple, efficient andinexpensive process for incorporating carbon dioxide and othercombustion gases, which are generated from stationary sources likecoal-fired power plants, into biomass while simultaneously removingthese gases from the environment.

Numerous proposals have been made to attempt to capture and removecarbon dioxide produced by fossil fuel combustion or from theatmosphere. These include using molecular sieves or solvents tophysically and chemical absorb carbon dioxide. Other methods include lowtemperature or cryogenic separation of carbon dioxide from air or othergases. Once carbon dioxide has been captured or separated in has beenproposed to transport in and store it in geological formations. However,these methods for capturing and storing carbon dioxide are logisticallycomplicated and expensive. Moreover such methods create newenvironmental risks including groundwater contamination oracidification, or geological instability including earthquakes. Suchmethods also create a new risk of leakage of the chemically orphysically captured or separated carbon dioxide back into theatmosphere. Physical or chemical adsorption or separation and storage ofcarbon dioxide also do not solve problems associated with other gaseouscomponents of combustion gas mixtures such as CO, NO_(x) and SO_(x).

In view of the above, the biological capture and fixation of carbondioxide and other combustion gases into more complex molecules usingmicroorganisms such as microalgae could offer a simple, convenient andeconomical way to capture carbon dioxide and other combustion gases.However, significant work is required to identify microalgae strains,culture media, and culture conditions suitable for efficient biofixationof carbon dioxide and other combustion gases. This is because differentmicroalgae strains have different biochemical properties and growthrates and because conventional media limit the rate at which amicroorganism can fix carbon dioxide or other combustion gases.

There is a significant need for a way to fix carbon dioxide and othercombustion gases into biomass, most preferably, valuable biomass such asnatural pigments. However, many conventional strains of microbes whichcan fix carbon dioxide have slow growth rates or produce little or nobiomass of significant value. For example, many strains of themicroalgae Haematococcus which can produce astaxanthin or other valuablenatural pigments have slow growth rates in conventional media. Theseslow growth rates both limit their ability to remove carbon dioxide andother combustion gases from the air as well as negatively impacting thevalue of biomass they produce.

In addition to fixing combustion gases into biomass, the microbialproduction of natural pigments such as astaxanthin could supply agrowing commercial market for these compounds which includepolyisoprenoids such as β-carotene, astaxanthin, and canthaxanthin.These pigments are receiving greater attention because they are oftenused in the food, nutrition, and cosmetics industries; PatriciaVeiga-Crespoet et al., Influence of culture conditions of Gordoniajacobaea MV-26 on canthaxanthin production, INTERNATIONAL MICROBIOLOGY,2005, 8, 1.

These pigments may be used for aquaculture of in animal feeds such aspoultry feed or in nutraceuticals. For example, astaxanthin is aderivative of beta-carotene that is used as a food additive and naturalcolorant to enhance the appearance of pale colored cultured fish andshellfish, such as salmonoid fish and shrimp. It has been reported tohave anti-inflammatory, anticancer and immunomodulatory properties andto confer numerous other health benefits for humans.

Haematococcus sp. often contain a high cellular content of astaxanthintypically ranging above 4% of its dry weight; Lee, Y. K., et al.,Accumulation of astaxanthin in Haematococcus lacustris (Chlorophyta).JOURNAL OF PHYCOLOGY. 1992, 3003, 575-577; Torzillo et al., JOURNAL OFAPPLIED PHYCOLOGY, 2003, 15: 127-136. The cost of the dried powder ofHaematococcus sp. is about 500 to $1,500/kg depending on its betacarotenoid and astaxanthin content; the price of purified astaxanthincan range upwards from about $70,000/kg.

Haematococcus is found world widely and its natural habitats arecharacterized by their unstable temporary conditions, which occur insmall rock pools, water holes, bird baths, and other small natural orartificial bodies of water. Haematococcus sp. are unicellular greenmicroalga with two flagella. They exhibit two types of cell morphologydepending on environmental conditions. Under optimal growth conditionsthe cells are green and vegetative, proliferative, and capable ofactively swimming using their flagella. However, under unfavorableconditions, the green vegetative cells drastically increase in volume,cease to be motile, and enter a resting stage to form cyst.

The green vegetative cells of Haematococcus are primarily composed ofcarotenoids which can contain 75-80 wt. % lutein, 10-20 wt. %β-carotene, as well as violaxanthin, neoxanthin, zeaxanthin, andchlorophyll a and b; Shah, M. M. R., et al., Astaxanthin-producing greenmicroalga Haematococcus pluvialis: From single cell to high valuecommercial products. FRONT. PLANT SCI. 2016, 7, 531.

Typically astaxanthin production from Haematococcus is achieved througha two-stage culture comprising a vegetative (green) stage andaplanospore (red) stage. However, vegetative cultivation ofHaematococcus is problematic due to slow growth rates, low celldensities, and susceptibility to contamination. Arrest of the growth ofHaematococcus has been attributed to various factors including theproduction of immotile cells at a pH above 9.0, which can occur when aculture generates significant amounts of ammonia; conversion ofvegetative motile cells into cysts after 4-6 days in conventional media;and release of cellular debris and blockade of the Haematococcus outermembrane by cellular debris. Unfortunately, while Haematococcus sp. canproduce valuable biomass they lack the growth rates necessary toefficiently metabolize large quantities of carbon dioxide such as thatin combustion or flue gases from coal-fired plants.

While many efforts have been made to boost the production of biomassfrom microalgae by using different media formulations, different kindsof vitamins, variation of light intensity or by use of mixed cultures ofdifferent algae, none have resulted in significant increases inproduction of algal biomass. Further, while conventional fed-batchmicroalgae culture can provide nutrients to facilitate continuedmicroalga growth, it does not remove chemical inhibitors of microalgagrowth, such as free fatty acids like EPA. This results in the cessationof growth of the microalgae cultured followed by its gradualdeterioration.

Apart from free fatty acid inhibitors, other culture components caninhibit microalga growth including debris generated from aging cellwalls and broken cells. The inventors found that these materials inhibitmicroalga growth by favoring the formation of large cell aggregateswhich sequester nutrients needed for active growth of Haematococcus andother microalgae and which diminish the ability of microalgae to adsorblight.

Other factors which impair or inhibit the growth of Haematococcus andthe production of valuable biomass include the presence of contaminatingmicroalgae or other microorganisms and sensitivity of microalgae toculture conditions, such as the source, intensity and light providedduring cultivation

In view of the problems described above, the inventors sought toformulate a culture medium and conditions for cultivation of selectHaematococcus microalgae strains that avoid the problems withconventional Haematococcus strains, culture media and culture methods inorder to enhance biofixation of combustion gases as well as yields ofvaluable biomass.

BRIEF SUMMARY OF THE INVENTION

In view of the limitations of conventional microalgae strains and thoseof conventional culture media and culture methods, the inventorsselected and cultured in vitro a new strain of microalgae designatedHaematococcus sp. KAU-01, formulated a new culture medium for thisstrain, and developed a new methods for mass culture for this strainthat inhibit contamination by other microorganisms, removes inhibitorsof microalgae growth, avoid undesired turbidity, and producecommercially valuable biomass using greenhouse gases and other productsof combustion thereby removing these gases as environmental pollutants.This high passage number strain has become stable after several passageswhere more than 80% of the cells died and has subsequently been viablystored. It has been designated Haematococcus sp., KAU-01. It may exhibitgenetic and epigenetic changes compared to the original isolate due torepeated passage and selection in vitro.

In conjunction with the selection of the microalgae Haematococcus sp.KAU-01, the inventors developed a new culture medium containing amixture of both micro- and macronutrients to cultivate this strain. Thisculture medium contains calcium nitrate in an amount that facilitatesthe growth and biomass production by Haematococcus sp. KAU-01 whileavoiding undesirable turbidity in the medium especially at high pH.

Another approach developed and used by the inventors to increase theefficiency of microalgae biomass production is the use of a filtersystem to remove inhibitors of microalgae growth without removing viablecells. One example of such a system is one that uses a bag net filterhaving a 5 μm mesh size through which spent culture medium containinginhibitors is discharged without loss of microalgae cells, followed bythe replacement of spent culture medium with fresh medium not containingthe inhibitors or particulate matter.

To provide a simple and more economical process of culturing ofHaematococcus sp. KAU-01, the inventors developed culture method thatdid not require sterilization of tap water or use of sterile distilledwater. They found that contaminating microorganisms could be controlledduring a bulk culture by acidifying culture water to a pH below 3.0 tokill microorganisms by acidic hydrolysis prior to adjusting the mediumto a more neutral pH and inoculating Haematococcus sp. KAU-01. This steppermits the use of unsterilized fresh or sea water and avoids thecomplexities and costs of autoclaving or filtering water prior to itsuse for mass or industrial-scale culture.

Embodiments of this technology include, but are not limited to thefollowing.

One aspect of this technology is a Haematococcus sp. that isHaematococcus sp. KAU-01 deposited under Patent Deposit NumberPTA-127272 or an engineered variant thereof that has genomic orribosomal DNA at least 99% identical to that of Haematococcus sp.KAU-01. Ribosomal DNA includes 18S and 26S rRNA gene sequence data.

In one embodiment, the Haematococcus sp. consists of Haematococcus sp.KAU-01, a subculture thereof, or a subculture that has been passaged invitro 10, 20, 30, 40, 50 or more times. Preferably, Haematococcus sp.KAU-01 is passaged in AAHKAU medium.

In another embodiment, the Haematococcus sp consists of a geneticallyengineered variant of Haematococcus sp. KAU-01 that compared to thedeposited strain of Haematococcus sp. KAU-01 that has been altered tohave at least one mutation to its DNA and/or that has been transformedwith an exogenous polynucleotide. These modifications include pointmutations and other alterations in the DNA sequence of Haematococcus sp.KAU-01 or its chloroplasts.

Methods and vectors for genetic modification of Haematococcus sp. KAU-01are known in the art and are incorporated by reference to Saei, A. A. etal., Haematococcus as a promising cell factory to produce recombinantpharmaceutical proteins MOLECULAR BIOLOGY REPORTS, 2012, 39, 9931-9939.In related embodiments, Haematococcus sp. KAU-01 may be used as a hostcell for the recombinant expression of vaccine proteins, enzymes, andother valuable proteins.

Haematococcus sp. KAU-01 may be modified to increase its stability byreducing epigenetic changes in its genome. Methods for epigeneticstabilization are known and are incorporated by reference to J. E. Pais& R. Spreafico. Avoiding epigenetic silencing of exogenous nucleic acidin algae, US20200190485A1, 2020. Thus, one embodiment of the inventionis directed to Haematococcus sp. KAU-01 that has been modified to bymutating or attenuating the methyltransferase (MTase) as well as othergenes involved in epigenetic modification of Haematococcus. For example,one embodiment is directed to a mutant or modified Haematococcus sp.KAU-01 comprising a mutated or attenuated gene encoding a polypeptidehaving a CHG DNA methyltransferase activity, wherein the mutant ormodified Haematococcus sp. KAU-01 has reduced CHG DNA methylation ascompared to Haematococcus sp. KAU-01.

Methods for chloroplast modification are known in the art and areincorporated by reference to Gutiérrez, C. L., et al., Chloroplastgenetic tool for the green microalgae Haematococcus pluvialis(Chlorophyceae, Volvocales), J. PHYCOLOGY, 2012, 48(4):976-983.

In another embodiment, the Haematococcus sp consists of anepigenetically modified or engineered variant of Haematococcus sp.KAU-01, Epigenetic modifications may be introduced by repeated passageand growth of Haematococcus sp. KAU-01 in vitro, such as passage orgrowth under stressful conditions, such as in nutrient depleted mediumor at lower or higher temperatures than 25 to 30° C. such as at 15, 20to <25° C. or >30, 35 to 40° C., in particular concentrations ofspecific nutrients or medium components, in medium exposed to combustiongases, under reduced or enhanced lighting compared to lightingconditions disclosed herein, under UV or chemical exposure, orcultivation at temperatures above or below room temperature or at highpH above 8, 9 or 10.

Another aspect of this technology is a method for producing biomasscomprising culturing Haematococcus sp. in a culture medium in thepresence of light, wherein the light may be present intermittently,periodically or continuously. In some embodiments, the light is natural,reflected, filtered, focused, or concentrated sunlight.

In other embodiments, the culture medium contains at least one materialcomprising a product obtained by combustion which can be incorporatedinto a gas, liquid or solid prior to incorporating it into the culturemedium.

The material, such as a combustion or greenhouse gas, may be obtained bycombustion of coal and in some embodiments is obtained by combustion ofcoal from or via a flue gas scrubber. This material usually comprisescarbon dioxide but may also constitute carbon monoxide, a nitrogenoxide, a sulfur oxide, a volatile organic compound. In some embodiments,the material may comprise a heavy metal present in the material prior toincorporating it into the culture medium. Preferably, undesirable ortoxic components like heavy metals are removed from the material priorto contacting it with a culture medium containing Haematococcus sp.KAU-01, In some embodiments of this method the Haematococcus sp. KAU-01is cultured by a fed batch method. For example, culturing can comprisefed-batch culturing wherein each feeding of the batch comprisesreplacing from 5, 10, 20, 30, 40, 50 to >50% of a spent culture mediumwith fresh culture medium and, optionally, adjusting the pH before,during or after feeding to range from 6 to 8.

In a preferred embodiment of a method for cultivating Haematococcus sp.KAU-01 the culture medium comprises AAHKAU medium. This medium containsthe following components given in mg/L and μl/L:

-   -   375.00 mg/L NaNO₃,    -   75.00 mg/L KNO₃,    -   25.00 mg/L Ca(NO₃)₂,    -   55.00 mg/L Mg(NO₃)₂·6H₂O,    -   10.00 mg/L K₂SO₄,    -   45.00 mg/L K₂HPO₄,    -   40.00 mg/L KH₂PO₄,    -   23.75 mg/L MgSO₄·7H₂O    -   1.76 mg/L urea,    -   65 μL/L HNO₃,    -   15 μL/L H₃PO₄;    -   3.50 mg/L FeCl₃·6H₂O,    -   1.00 mg/L H₃BO₃,    -   0.25 mg/L Co(NO₃)₂·6H₂O,    -   0.10 mg/L K₂Cr₂O₇,    -   0.10 mg/L CuSO₄·5H₂O,    -   0.25 mg/L MnSO₄·H₂O,    -   0.75 mg/L ZnSO₄·6H₂O,    -   0.25 mg/L (NH₄)₆Mo₇O₂₄·4H₂O    -   1.00 mg/L Na₂-EDTA and    -   1.00 μl/L HCl; and a mixture of vitamins.

Typically the medium also contains a vitamin solution xxx

AAHKAU medium (1×) may be formulated from stock solutions A, B and C.

Stock solution A (1000×) contains:

-   -   375.00 g/L NaNO₃,    -   75.00 g/L KNO₃,    -   25.00 g/L Ca(NO₃)₂,    -   55.00 g/L Mg(NO₃)₂·6H₂O,    -   10.00 g/L K₂SO₄,    -   45.00 g/L K₂HPO₄,    -   40.00 g/L KH₂PO₄,    -   23.75 g/L MgSO₄·7H₂O    -   1.76 g/L urea,    -   65 ml/L HNO₃, and    -   15 ml/L H₃PO₄;

Stock solution B (10,000×) contains:

-   -   35.00 g/L FeCl₃·6H₂O,    -   10.00 g/L H₃BO₃,    -   2.50 g/L Co(NO₃)₂·6H₂O,    -   1.00 g/L K₂Cr₂O₇,    -   1.00 g/L CuSO₄·5H₂O,    -   2.50 g/L MnSO₄·H₂O,    -   7.50 g/L ZnSO₄·6H₂O,    -   2.50 g/L (NH₄)₆Mo₇O₂₄·4H₂O    -   10.00 g/L Na₂-EDTA and    -   10.00 ml/L HCl.

Stock solution C (10,000×) typically contains 10 g/L thiamine, 0.03g/L+D-biotin, and 0.001 g/L cyanocobalamin.

Typically, 1 ml of the 1000× stock solution 1, 0.1 ml of the 10,000×stock solution B, and 0.1 ml of the 10,000× stock solution B are dilutedin water up to 1 L to form 1×AAHKAU medium. However, other amounts ofthese stock solutions may be used to produce mediums with a lower orhigher concentration of particular components. The exact amounts of eachingredient in the stock solutions A, B and/or C or in the 1×AAHKAUpreparation from stock solutions may vary by ±1, 2, 3, 4, 5, 6, 7, 8, 9or 10%.

In the formulations above, in the Tables, and elsewhere herein, theconcentrations of the ingredients forming a medium may vary by ±1, 2, 5,or 10% by weight or volume.

In a preferred embodiment of this method the Haematococcus sp.comprises, consists essentially of, or consists of Haematococcus sp.KAU-01, a subculture thereof, or a genetically or epigeneticallymodified variant thereof.

In some embodiments of this method, biomass that is produced by theHaematococcus culture is recovered by filtration, centrifugation and/orsedimentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Variation in maximum specific growth rate (u_(max) d⁻¹) andgrowth curves with cell abundance (×10⁵ cells mL⁻¹) of Haematococcus sp.KAU cultured in AAHKAU medium within ‘A’ culture group. A1: continuousaeration with normal air; A2: continuous aeration with normal air, pHwas maintained at pH 7.5 by injecting amounts of stock solutions A andB; A3: continuous aeration with normal air, 20% media replacement onalternate days.

FIG. 2 . Variation in maximum specific growth rate (u_(max) d⁻¹) andgrowth curve with cell abundance (×10⁵ cells mL⁻¹) of Haematococcus sp.KAU cultured in AAHKAU medium within ‘B’ culture group. B1 normalaeration+15% CO₂; B2: normal aeration+15% CO₂, pH maintained at pH 7.5and additionally pH was maintained 7.5 by injecting of stock solution Aand B on alternative day; B3: normal aeration+15% CO₂, 20% mediareplacement on alternate days.

FIG. 3 . Variation in maximum specific growth rate (u_(max) d⁻¹) andgrowth curve with cell abundance (×10⁵ cells mL⁻¹) of Haematococcus sp.KAU cultured in AAAHKAU medium within “C” culture group: C1 normalaeration+15% CO₂+CO 5%+NO 1%+NO₂ 1%+N₂ balance; C2: normal aeration+15%CO₂+CO 5%+NO 1%+NO₂ 1%+N₂ balance, pH maintained at pH 7.5, andadditionally pH was maintained 7.5 by injecting of stock solutions A andB on alternate days; C3: normal aeration+15% CO₂+CO 5%+NO 1%+NO₂ 1%+N₂balance, 20% media replacement on alternate days.

FIG. 4 . Variation of biomass (gL⁻¹) production of Haematococcus sp. KAUcultured in AAHKAU medium in A culture group.

FIG. 5 . Variation of biomass (gL⁻¹) production of Haematococcus sp. KAUcultured in AAHKAU medium in B culture group.

FIG. 6 . Variation of biomass (gL⁻¹) production of Haematococcus sp. KAUcultured in AAHKAU medium in C culture group.

FIG. 7 . Amount of dry particulate matter (cellular debris) from brokencells and cell walls during culture of Haematococcus sp. KAU in culturesubgroups A3, B3 and C3 in which 20% of culture medium was replaced onalternate days after day 4 of culture.

FIGS. 8A-8B. Maps of sample collection area for Haematococcus sp.KAU-01.

FIGS. 8C-8D. Terrain of sample collection area at Green Valley, Jeddah,the Kingdom of Saudi Arabia.

FIG. 9 . Green vegetative cell with (a) two flagella, (b) trilaminarsheath, (c) cytoplasmic strands and (e) pyrenoids as observed underinverted light microscope.

FIGS. 10A-10F. Life cycle from green, vegetative motile form tonon-motile aplanospore.

FIG. 10A: shows green, motile vegetative flagellated cells.

FIG. 10B shows large aplanospores with red astaxanthin at the center andspreading towards a circle and green chlorophyll a around peripheries.

FIG. 10C shows fully red color aplanospore.

FIGS. 10D and 10E depict asexual reproduction of aplanospore.

FIG. 10F shows daughter cell are coming out by breaking aplanospore cellwall with little astaxanthin.

FIG. 11A depicts flagellated cells of Haematococcus sp. KAU-01.

FIG. 11B shows cell walls broken or lysed due to lack of Ca-salts.

FIG. 11C shows cells without breaking of cell wall after graduallyincreasing Ca-salt concentration to 40 mgL⁻¹.

FIG. 11D depicts red cysts without lysis of cell.

FIG. 12A. Growth performance and biomass (dry wt. gL⁻¹) production ofHaematococcus sp. KAU-01 grown in seven different culture media ascomparable to AAHKAU medium.

FIG. 12B. Growth performance and cell abundance of Haematococcus sp.KAU-01 grown in seven different culture media as comparable to AAHKAUmedium.

FIGS. 13A-13F. Growth and dry biomass production of Haematococcus sp.KAU, culture in square design combinations of A and B stock solutions.See Tables 2, 3 and 4 below for media formulations for A1-A5 and B1-B5.

FIG. 13A. Dry biomass produced by culture in major nutrientconcentrations A1 in combination with minor nutrient concentrationsB1-B5.

FIG. 13B. Dry biomass produced by culture in major nutrientconcentrations A2 in combination with minor nutrient concentrationsB1-B5.

FIG. 13C. Dry biomass produced by culture in major nutrientconcentrations A3 in combination with minor nutrient concentrationsB1-B5. Combination A3B3 provided superior results compared to other A3combinations.

FIG. 13D. Dry biomass produced by culture in major nutrientconcentrations A4 in combination with minor nutrient concentrationsB1-B5.

FIG. 13E. Dry biomass produced by culture in major nutrientconcentrations A5 in combination with minor nutrient concentrationsB1-B5.

FIG. 13F. Dry biomass produced by culture in major nutrientconcentrations A3 in combination with minor nutrient concentration B3(“A3B3”) compared to conventional media OHM, RM, Basal and HK.Combination A3B3 provides superior results compared to all comparativemedia.

FIG. 14A. Variation of biomass production of Haematococcus sp. KAU-01grown in comparative culture media OHM, Basal, RM, HK and AAHKAU (A3B3)1 (sterile filtered, distilled water) and AAHKAU (A3B3) 2 (non-steriletap water). AAHKAU-1 was prepared autoclaved sterile distilled water andAAHKAU-2 was prepared using municipal supplied water which has been notbeen directly acid treated, but which was made acidic after dilution ofstock solution of ‘A’ and ‘B’ as both solution contain acids.

FIG. 14B. Variation in maximum specific growth rate (umax d⁻¹) andgrowth curve with cell abundance (Cells L⁻¹) of Haematococcus sp. KAU-01cultured in different culture media including newly prepared AAHKAU(A3B3) medium.

DETAILED DESCRIPTION OF THE INVENTION

Haematococcus is a genus of algae in the family Haematococcaceae. Theterm Haematococcus sp. describes an unspecified species in the genusHaematococcus and Haematococcus sp. KAU-01 describes the particulardeposited unspecified species isolated from a hot, tropical climate,cultivated or selected in vitro, and characterized by the inventors. Incontrast to the native isolate, this strain can exhibit genetic orepigenetic changes that permit it to efficiently grow outside itsnatural environment, fix combustion gases, and produce valuable biomass.Haematococcus sp. KAU-01 is a stable monostrain which has been depositedunder the terms of the Budapest Treaty at the ATCC Patent Depository,10108 University Boulevard, Manassas, Va. 20110 under Patent DepositNumber PTA-12772.

Haematococcus sp. KAU-01 was isolated away from other microalgaeidentified at Green Valley of Jeddah KSA including away from microalgaewhich belong to Chlorophyta such as Pediastrum sp., Scenedesmus sp., andVolvox sp. These other kinds of Chlorophyta differ from Haematococcussp. KAU-01 as evident from their morphological characteristics. Based onits growth and metabolic properties, the inventors believe thatsignificant phylogenetic differences distinguish Haematococcus sp.KAU-01 from other Haematococcus sp. such as H. pluvialis, H. capensis,H. carocellus, H. lacustris, and H. zimbabwiensis.

The parent isolated of the Haematococcus sp, which was later passagedand deposited as Haematococcus sp. KAU-01, was isolated from a naturalsample using several steps. Initially, free swimming cells werecollected using mouth sucking micro-pipetting and innoculated culturedin F/2 medium. Soon after inoculation, it was found that some cell wallswere broken and the cellular materials were coming out resulting thedeath of the cells. Subsequently, the natural sample was filtered ontowhatman (GF/F) which was kept at room temperature for about four days todry. Thereafter, the whole filter paper was again sunk in the F/2culture medium. Free swimming cells were again isolated by mouth suckingmicropippetting.

Agar culture of the free swimming cells was done to get a mono-strain,the growth of which was then evaluated on several different culturemedia: BG11, MBG11, OHM, Basal, RM, HK, and MCM, see Table 1.Unfortunately, it was found that cell growth ceased after only 4-6 dayswhen the free swimming cells were inoculated and cultured in thesemedia. Therefore, it was decided to formulate a new culture medium forthis species.

Several trial and error studies were conducted using novel media. Cellsgrown in these media were continuously observed under the microscope.Observations were made of cell division, cell movement, cyst formation,and regeneration from the cysts, as well as culture turbidity andsedimentation. As disclosed herein, a medium designated AAHKAU mediumwas developed using this process specially formulated for cultivatingthe isolated Haematococcus sp. KAU-01.

Microalgae including Haematococcus may be mutagenized by proceduresknown in the art including by exposure to mutagens such as UV light,ethyl methane sulphonate (EMS) and 1-methyl 3-nitro 1-nitrosoguanidine(NTG); Kamath, et al., BIORESOURCE TECHNOLOGY, 2008, 99, (18), 8667-8673(incorporated by reference). Such methods may be used to mutateHaematococcus sp. KAU-01.

Haematococcus and other microalgae may be genetically engineered andtransformed with exogenous polynucleotides by other methods known in theart; Sharon-Gojman, et al., ALGAL RESEARCH, 2015, 10, 8-15 orSteinbrenner, et al., APPL. ENVIRON. MICROBIOL. 2006, 72(12): 7477-7484(both incorporated by reference). Such methods are used to transform orotherwise genetically modify Haematococcus sp., such as Haematococcussp. KAU-01.

In some embodiments of the methods disclosed herein, a natural isolateor subculture thereof of a Haematococcus sp. is used. In otherembodiments, Haematococcus sp. KAU-01 is further engineered andmodified, mutated, or epigenetically or genetically engineered.

Modified variants of Haematococcus sp. KAU-01 may have one or morecoding sequences, genes or polypeptides with alterations, such asdeletions, substitutions or insertions of nucleotides or amino acidresidues. Generally, related strains will have genomic DNA or one ormore ribosomal DNA genes 95, 96, 97, 96, 99, 99.5, 99.9, <100 or 100%identical or similar to that of a parent or natural isolate of aHaematococcus sp., such as Haematococcus sp. KAU-01.

BLASTN may be used to identify a polynucleotide sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100%sequence identity to a reference polynucleotide such as a polynucleotideof Haematococcus sp. KAU-01. A representative BLASTN setting modified tofind highly similar sequences uses an Expect Threshold of 10 and aWordsize of 28, max matches in query range of 0, match/mismatch scoresof 1/−2, and linear gap cost. Low complexity regions may be filtered ormasked. Default settings of a Standard Nucleotide BLAST are described byand incorporated by reference to hypertext transfer protocolsecure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Oct. 13, 2020).

A mutant or variant form of Haematococcus sp. KAU-01 may becharacterized by expression of one or more mutant proteins that differfrom a corresponding protein of Haematococcus sp. KAU-01 or by geneticor epigenetic modifications to its nucleic acids.

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100%sequence identity, or similarity to a reference amino acid, such as amutant Haematococcus sp. KAU-01 amino acid sequence, using a similaritymatrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be usedfor closely related sequences, BLOSUM62 for midrange sequences, andBLOSUM80 for more distantly related sequences. Unless otherwiseindicated a similarity score will be based on use of BLOSUM62. WhenBLASTP is used, the percent similarity is based on the BLASTP positivesscore and the percent sequence identity is based on the BLASTPidentities score. BLASTP “Identities” shows the number and fraction oftotal residues in the high scoring sequence pairs which are identical;and BLASTP “Positives” shows the number and fraction of residues forwhich the alignment scores have positive values and which are similar toeach other.

Amino acid sequences having these degrees of identity or similarity orany intermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Other default settings for BLASTPare described by and incorporated by reference to the disclosureavailable at: hypertext transfer protocolsecure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Oct. 13, 2020).

In some embodiments, Haematococcus sp. KAU-01 is further modified ormutated, for example, by chemical or radiological mutation or by geneticengineering and recombinant DNA techniques. A modified form ofHaematococcus may have one or more epigenetic changes to its DNA, suchas a variant methylation or hydroxymethylation pattern in its genomicDNA, a difference in histone methylation, or difference in microRNAexpression, compared to an otherwise identical isolate. Epigeneticvariants are those having a heritable phenotype change that does notinvolve alterations in its DNA sequence.

Such modifications may be made to improve growth or biomass productionunder particular culture conditions, to protect it from contamination byother microorganisms, for example, by making it resistant to one or moreantibiotics, or to improve its functional properties such as its growthrate or ability to produce higher densities of biomass under particularculture conditions, its ability to form or not form a biofilm, itsability to adhere or not adhere to a substrate such as plastic, glass,ceramic, or metal, or its ability to absorb light within a specificrange of wavelengths, or to boost its ability to produce valuablebiological materials, such as fatty acids or astaxanthin or hydrogen(hypertext transfer protocolsecure://newatlas.com/algae-hydrogen-production-boost-tau/45831/). Sucha modified microalgae may also be mutated or engineered to preventproduction of one or more of such biological materials that interferewith growth of the microalgae or production of biomass. Modificationsinclude induction of auxotrophy for one or more molecules made by theunmodified strain or the incorporation of expression control sequencessuch as repressible promoters into the genome or episomes of themicroalgae.

Greenhouse gases absorb and emit radiation within the thermal infraredrange and include water vapor, carbon dioxide, methane, nitrous oxide,and ozone.

Flue gases. Flue gases are produced when natural gas, fuel oil, coal,wood, cellulose wastes (e.g., grass, palm fronds, paper, cardboard,etc.) or any other carbon based fuel is combusted in an industrialfurnace, a steam generator in a fossil fuel power plant or othercombustion sources. Typically, flue gases consist of mostly nitrogen(typically more than two-thirds) derived from the combustion of air,carbon dioxide (CO₂), and water vapor as well as excess oxygen which maybe derived from the combustion air and contain small percentages ofvarious of pollutants, such as matter like soot, carbon monoxide,nitrogen oxides, sulfur oxides, volatile organic compounds, andhydrocarbons; see Fossil fuel combustion flue gases, Milton R. Beychok,ENCYCLOPEDIA OF EARTH, 2012 (incorporated by reference). Volatileorganic compounds includes those emitted by burning coal such asaldehydes (formaldehyde and acetaldehyde), aliphatic and aromatichydrocarbons (toluene, xylenes, ethylbenzene and benzene) andchlorinated hydrocarbons (tetrachloroethene); see Garcia, et al.,ATMOSPHERIC ENVIRONMENT. PART A. GENERAL TOPICS, 1992, 26(9), 1589-1597(incorporated by reference).

In some embodiments, one or more flue or greenhouse gases or pollutantsare adsorbed or dissolved into a growth medium for Haematococcus sp.KAU-01. For example, in some embodiments a culture medium is contactedwith or infused with a mixture of air with 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% (v/v)carbon dioxide or other greenhouse or flue gases. The medium may becontacted or infused with one or more gases at a temperature rangingfrom 0, 5, 10, 15, 20, 25 or 30° C. and/or at a pressure of 1 (14.7psi), 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3 or moreatmospheres. The resulting medium may contain microalgae or be added orfed to an ongoing culture of microalgae.

In one embodiment, coal power plant gases are taken from the coal powerplant chimney with pressure in to a reserve tank, held to adjust thetemperature to one similar to air temperature, and then the gases aresupplied to a Haematococcus culture so that the culture medium has a pHranging from about 7.0-8.0, preferably about pH 7.5, The gasesthemselves or other buffers may be used to adjust the culture medium pH.

Cooling and heating may be performed in a tank or other containerequipped with refrigeration or heaters and infusion conducted in apressurized tank or other container.

Temperature and pressure sensors, pumps or regulators may beincorporated into the tank as well as conduits or valves for deliveringthe temperature controlled or infused medium to feed the microalgae.

Fed-batch culture is an operational technique in biotechnologicalprocesses where one or more nutrients (substrates) are fed (supplied) toa bioreactor during cultivation and in which the product(s) remain inthe bioreactor until the end of the run. In some embodiments of theinvention Haematococcus sp. KAU-01 is cultivated in a fed-batch culturewhich provides fresh nutrients for its growth and which removes spentmedium containing growth inhibitors, metabolites, or cellular debristhat inhibit production or accumulation of biomass. Nutrients may besupplied to the fed-batch culture constantly or at a particular rate,such as by exponential feeding that is tied to the number of cells inthe culture. In some embodiments, this method will include fed-batchculture of Haematococcus sp. where each feeding involves replacing oradding (supplementing with) from 1, 2, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% by volume of thespent culture medium with fresh culture medium. Culture medium may bereplaced every 12, 24 or 48 hours (or any intermediate period).

Advantageously, culture medium may be replaced every other day after aninitial culture period of 1, 2, 3, 4, 5 or 6 days. The temperature maybe adjusted to fall within the range of 15, 20, 25, to 30° C. and pHadjusted before, during or after feeding for example, by adding acid,base or a buffer to bring the culture pH to 5, 5.5, 6, 6.5, 6.6, 6.7,6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 or 8 or any intermediate valuewithin this range. In some embodiments, up to 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 95, <100 or 100% of EPA or cellular debris are removed ordiluted below their initial concentration, for example, by filtration ofspent medium and replacement with fresh medium not containing thesecontaminants.

The rate of microalgae growth may be controlled by adjusting thetemperature or pH of the culture or by reducing or increasing one ormore nutrients or growth substrates, including an amount of one or moreflue gases, for the microalgae.

In some embodiments, following fed-batch culture, the microalgae areharvested by sedimentation, filtration, or centrifugation. Microalgaemay be harvested at one or more of their life cycle stages includeduring vegetative cell growth, encystment, maturation or germination.

Harvested microalgae may be used to make biofuel, nutraceuticals such asomega three fatty acids, glycoproteins, pigments or astaxanthin, or asanimal or aquaculture feeds. Biological products made by microalga suchas Haematococcus include beta-carotene, astaxanthin, canthaxanthin,lutein, other polyisoprenoids, EPA and other fatty acids. These may beharvested or recovered at a point in the alga lifecycle where they aremaximally expressed or at a point where their purity is high at a timewhen little degradation or chemical transformation of the desiredproduct has occurred, or when it is easy to isolate them from otheralgae components, for example, beta-carotene and lutein may be harvestedwhen an algae is in a green vegetative state and astaxanthin from redcysts.

Bioreactor. Typically a bioreactor will include a container, such as atank and a light source, where the tank contains a culture medium anddispersed algal cells such as Haematococcus sp. KAU-01 cells. The algalcell culture may have a concentration of greater than 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0 or >2.0 g/l in the culturemedium.

It may have a paddle or other kind of agitator or mixer to keep thecells in suspension. Representative bioreactors or culture systemsinclude circulating open pond, open racing pond, bubble column, airliftreactor, annular reactor, stirred tank reactor, flat panel (plate)reactor, hemispherical biodome, or simple horizontal tumbular reactorany of which may accommodate exposure of the algae to light by providingdirect light exposure or exposure to light through a transparent ortranslucent or illuminated panel or container. A bioreactor may containone or more mixing devices including, but not limited to, paddlewheels,gas sparging, and mechanical stirrers, to help expose cells toillumination and prevent them from settling to the bottom of abioreactor. Bioreactors may include inlets (for fresh medium components)or outlets (to let out spent medium components or to recover algae). Insome embodiments, the inlets and outlets are valved to regulate the flowof components. A bioreactor may also be equipped with filters or otherseparation devices that retain algae and pass spent culture mediumcomponents as well as cellular debris out of the culture medium.

A photobioreactor (PBR) is a bioreactor which incorporates a lightsource and may be used to cultivate Haematococcus sp. KAU-01 cells.Virtually any translucent container may be called a PBR and in someembodiments the PBR is a tank, a polyethylene or other clear ortranslucent plastic sleeve or gag or clear or translucent tubes, such asglass or plastic tubes. This term typically describes a closed system asopposed to an open tank or pond. A closed PBR system generally providesall the nutrients and micronutrients necessary for algae growth. A PBRcan operate in “batch mode”, which involves restocking the reactor aftereach harvest, but it is also possible to grow and harvest continuously.Continuous operation requires precise control of all elements to preventimmediate collapse. The grower typically provides sterilized water,nutrients, air, and carbon dioxide or other greenhouse or flue gases ata predetermined rate.

Algae grown in the log phase is generally of higher nutrient content orbetter at removing or remediating flue or greenhouse gases than oldsenescent algae. In some embodiments of the invention, Haematococcus sp.KAU-01 cells are grown in a photoreactor using procedures and/orequipment that permit removal of growth inhibitory substances. In otherembodiments, Haematococcus sp. KAU-01 may be grown under condition whereEPA and cellular debris are continuously removed, for example, bygrowing it in flat transparent porous bags that retain viableHaematococcus sp. KAU-01 but permit outward diffusion of EPA andcellular debris or that permit sedimentation of cellular debris. Theseporous bags may be continuously, periodically or intermittently suppliedwith fresh culture medium.

Algal culture is the culturing of algae in ponds or other resources.Other modes of algae culture are described by and incorporated byreference to Borowitzka, Commercial production of microalgae: ponds,tanks, and fermenters, PROGRESS IN INDUSTRIAL MICROBIOLOGY, 1999, 35,313-321. In some embodiments of the invention, Haematococcus sp. KAU-01cells are grown in algal culture using procedures and/or equipment thatpermit removal of growth inhibitory substances.

Filtration. Spent medium which may contain growth inhibitory substancesis typically removed by filtration through one or more filters thatretain viable Haematococcus sp. KAU-01 cells, but which permit passageof growth inhibitory substances such as eicosapentaenoic acid (EPA) andcellular debris. These cells may range in particle size from about 5 to25 microns and some representative filter mesh sizes range from about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25 microns. As shown herein a filter bag or phytoplanktonnet mesh size of about 5 or 10 microns is suitable for retaining viableHaematococcus sp. KAU-01 cells. Other conventional or commerciallyavailable algae filters or scrubbers may be used. In some embodiments,other separation modes may be used including centrifugation orsedimentation to separate or partially separate viable cells from otherculture components.

Other embodiments of the invention include, but are not limited to thefollowing.

One embodiment of the invention is a wild type (unmodified),epigenetically modified, chemically modified, irradiated, mutated, orgenetically engineered variant of Haematococcus sp. KAU-01 that hasgenomic DNA at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9,<100 or 100% identical to Haematococcus sp. KAU-01. A mutated orgenetically engineered variant may contain 1, 5, 10, 20, 50, 100, 200,500 or more deletions, substitutions or insertions of nucleotides intothe genomic DNA or 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100 or moredeletions, substitutions or insertions into one or more proteinsexpressed by the microalgae. In some embodiments, this microalgae is astrain of Haematococcus other than Haematococcus sp. KAU-01 in admixturewith other microalgae or microorganisms or as it may be found in nature.In some embodiments, the microalgae will be a genetically engineeredvariant of Haematococcus sp. KAU-01 that has been transformed with atleast one exogenous polynucleotide, for example, by transformation withone or more plasmids or transposons that express antibiotic resistanceor other selectable markers, which express enzymes which enhancemicroalgae growth or biomass production, or which contain controlsequences that modulate (decrease, stabilize or enhance) microalgaegrowth or biomass production.

A Haematococcus sp. as isolated from nature may be further modified, forexample, by chemical or radiological mutation or by genetic engineeringsuch as by transformation with an exogenous polynucleotide sequence froma different type of organisms, such as a plant, microalgae, differenttype or strain of Haematococcus, animal, plant, fungus, or othereukaryotic or prokaryotic organisms, or by an artificial polynucleotidesequence. Exogenous polynucleotide sequences include detectable markersor sequences coding for chemical or antibiotic resistance, reporter,promoter, termination control sequence, and other expression controlsequences, sequences coding biosynthetic enzymes or enhancing orotherwise modulating expression of astaxanthin, carotenoids or otherchemical products produced by a microalgae. The sequence of an exogenousgene may be codon-modified to match it to that of a Haematococcus hostcell.

Another embodiment of the invention is method for producing biomasscomprising culturing in the presence of light and in a suitable mediumHaematococcus sp. KAU-01 or a Haematococcus strain that has genomic DNAat least 80, 85, 90, 95, 96, 97, 98, 99, 99, 99.5, 99.9, <100 or 100%identical to Haematococcus sp. KAU-01 thereby producing biomass. In thismethod the light may be natural, reflected, filtered, focused orconcentrated sunlight or, alternatively, artificial light or a mixtureof natural and artificial light having a wavelength suitable for cultureof Haematococcus sp. KAU-01. The wavelength of the used to cultivatemicroalgae is not particularly limited as long as it can be adsorbed andused by the microalgae to product biomass. Visible light having awavelength ranging from 380, 400, 450, 500, 550, 600, 650, 700, 750 to780 nm may be used. The amount or intensity of the light used tocultivate the microalgae is not particularly limited as long as it issufficient for the microalgae to produce biomass. For example,respective photosynthetic photon flux densities (PPFD) in the vicinityof a light irradiation surface of the culture solution may range from 5μmol/m²/s to 200 μmol/m²/s, preferably 10 μmol/m²/s to 100 μmol/m²/s,and more preferably 20 μmol/m²/s to 70 μmol/m²/s.

Illumination of the microalgae may be continuous, periodic orintermittent. Preferred illumination periods range from 1, 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22 or 24 hours and may be cyclic or non-cyclic.Haematococcus sp. KAU-01 is grown at a temperature suitable for itsgrowth or production of biomass, for example, at a temperature rangingfrom 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34 or 35° C.

In some embodiments Haematococcus sp. KAU-01 is cultured outside, forexample, in an outdoor circulating pool exposed to sunlight whichcontains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, 1.0, 1.1,1.2, 1.3, 1.4, 1.5 or 2× of the culture medium ingredients describedherein. Preferably, such culturing is performed at a pH ranging from7.0-8.0, advantageously at an adjusted pH of 7.5.

One or more greenhouse or flue gases may be incorporated into orotherwise contacted with the medium in this method. These may be thedirect products of combustion, such as coal, petroleum, natural gas,gasoline, diesel or other petrochemical combustion, or may be modified,mixed or processed prior to incorporation into a culture medium, forexample, by adsorption into scrubber gases, liquids or solids to form aderivative or by admixture with air or other gases. Combustion gasesinclude carbon dioxide, carbon monoxide, nitrogen oxide, sulfur oxides,and volatile organic compounds. Such gases in unpurified, partiallypurified or purified form may be pumped into or over or blown over themedium, or otherwise perfused or incorporated into the medium. In someembodiments products, such as those produced in a flue gas scrubber orcatalytic converter, may be incorporated into the culture medium.

In some embodiments, one or more contaminants, such as heavy metal,carbon, ash, or other products of combustion may be removed from orseparate from one or more of these combustion gases or their mixtures orderivatives prior to incorporation into a culture medium. Heavy metalsinclude arsenic, antimony, lead, zinc, manganese, nickel, copper, andchromium. Reductions ranging from <5%, 10, 20, 30, 40, 50, 60, 70, 80,90, 95, <100, or 100% of these metals may be made to lessen the impactof these metals on the growth of microalgae or the contamination ofbiomass produced by microalgae. Heavy metals may be captured and removedfrom combustion gases by methods known in the art include thosedescribed by Chen, et al., Science of the Total Environment 228 (1):67-77 (1999, incorporated by reference) or Li, et al., Fuel 186:714-725(2016, incorporated by reference).

In some embodiments, this method will include fed-batch culture ofHaematococcus sp. KAU-01 where each feeding involves replacing from 1,2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100% by volume of the spent culture medium with fresh culturemedium. The culture pH may be adjusted during, between or after afeeding, for example, by adding acid, base or a buffer to bring theculture pH to 5, 5.5, 6, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5 or 8 or any intermediate value within this range. Advantageouslyfeedings are performed using AAHKAU medium or an AAHKAU-like medium andperformed using Haematococcus sp. KAU-01 or a variant thereof whosegenomic DNA is least 80, 85, 90, 95, 96, 97, 98, 99, 99.5 or <100%identical to Haematococcus sp. KAU-01. In some embodiments, not mutatedor not-engineered Haematococcus sp. KAU-01 in other embodiments amodified mutated or engineered variant of Haematococcus sp. KAU-01 thatdiffers from the original isolate is cultivated.

In some embodiments, the biomass is recovered by filtration,centrifugation, and/or sedimentation of medium or other liquidcontaining the biomass. Biomass recovered from culture medium may befurther washed, rinsed, dried, concentrated or otherwise processed, forexample, to isolate a chemical component it contain such as a fattyacid, pigment, astaxanthin, or other nutraceutical product, to place itin a form for processing into biodiesel or other fuel, to place it in ausable form transport, or to put in into a form suitable as an animal oraquaculture feed.

In other embodiments, a species other than a Haematococcus sp. may becultured in AAHKAU medium, such microorganisms include Chlorophyta suchas Pediastrum sp. and Scencedesmus sp,

Another embodiment of the invention is a composition comprising (i) anartificial culture medium and (ii) Haematococcus sp. KAU-01 or a mutatedor genetically engineered variant of Haematococcus sp. KAU-01 that hasgenomic DNA at least 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, <100 or100% identical to Haematococcus sp. KAU-01. This composition may furtherinclude at least one greenhouse or gas resulting from the combustion ofat least one hydrocarbon such as a flue gas from a coal-fired powerplant. For example, the composition may contain one or more gases suchas carbon dioxide generated by combustion or oxidation of fuel sourcessuch as coal, petroleum, petrol (gas), diesel fuel, or alcohol or otherfuels that produce carbon dioxide or greenhouse gases when oxidized.

Another embodiment of the invention is directed to a culture mediumcomprising major nutrients in the following ranges:

187.50 to 562.50 mg/L NaNO₃, 37.50 to 112.50 mg/L KNO₃, 12.50 to 37.50mg/L Ca(NO₃)₂, 27.50 to 82.50 mg/L Mg(NO₃)₂.6H₂O, 22.50 to 67.50 mg/LK₂HPO₄, 20.00 to 60.00 mg/L KH₂PO₄, 5.00 to 15.00 mg/L K₂SO₄, 11.88 to35.63 mg/L MgSO₄.7H₂O, 0.88 to 2.63 mg/L urea, 32.50 to 97.50 μl/L HNO₃,7.50 to 22.50 μl/L H₃PO₄, and freshwater.

In typical embodiments, this culture medium further includes one or moreof the following micronutrients FeCl₃·6H₂O, H₃BO₃, Co(NO₃)₂·6H₂O,K₂Cr₂O₇, CuSO₄·5H₂O, MnSO₄·H₂O, ZnSO₄·6H₂O, Na₂-EDTA and HCl. In oneembodiment the micronutrients and vitamins fall within the followingranges 1.75 to 5.25 mg/L FeCl₃·6H₂O, 0.50 to 1.50 mg/L H₃BO₃, 0.13 to0.38 mg/L Co(NO₃)₂·6H₂O, 0.05 to 0.15 mg/L K₂Cr₂O₇, 0.05 to 0.15 mg/LCuSO₄·5H₂O, 0.13 to 0.38 mg/L MnSO₄·H₂O, 0.38 to 1.13 mg/L ZnSO₄·6H₂O,0.13 to 0.38 mg/L (NH₄)₆Mo₇O₂₄·4H₂O, 0.5 to 1.5 mg/L Na₂-EDTA, and 0.5to 1.5 μl/L HCl.

Preferably, the 1× medium further comprises 0.8 to 1.2 mg/L thiamine,0.0024 to 0.0036 mg/L+D-biotin, and 0.00008 to 0.00012 mg/Lcyanocobalamin.

The ranges herein include all intermediate subranges and values. Forhydrated compounds, these amounts include the equivalent molar amountsof anhydrous or other hydrates of the same compound.

One example of an advantageous medium formulation is 1×AAHKAU mediumsolution as a Haematococcus sp., KAU-01 culture medium which contains:

-   -   375.00 mg/L NaNO₃,    -   75.00 mg/L KNO₃,    -   25.00 mg/L Ca(NO₃)₂,    -   55.00 mg/L Mg(NO₃)₂·6H₂O,    -   10.00 mg/L K₂SO₄,    -   45.00 mg/L K₂HPO₄,    -   40.00 mg/L KH₂PO₄,    -   23.75 mg/L MgSO₄·7H₂O,    -   1.75 mg/L urea,    -   65.00 μL/L HNO₃,    -   15.00 μL/L H₃PO₄;    -   3.50 mg/L FeCl₃·6H₂O,    -   1.00 mg/L H₃BO₃,    -   0.25 mg/L Co(NO₃)₂·6H₂O,    -   0.10 mg/L K₂Cr₂O₇,    -   0.10 mg/L CuSO₄·5H₂O,    -   0.25 mg/L MnSO₄·H₂O,    -   0.75 mg/L ZnSO₄·6H₂O,    -   0.25 mg/L (NH₄)₆Mo₇O₂₄·4H₂O,    -   1.00 mg/L Na₂-EDTA,    -   1.00 μL/L HCl,    -   1.00 mg/L thiamine,    -   0.003 mg/L+D-biotin, and    -   0.0001 mg/L cyanocobalamin;    -   wherein each of said concentrations (mg/L) may vary 0.5, 1, 1.5,        2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6.0, 7.5, 8, 8.5, 9, 9.5, or        10%.

In embodiments where greenhouse or combustion gases or by products areincorporated into a culture medium, these additional components mayconstitute at least a part of the one or more of the ingredients ofAAHKAU medium or other culture medium disclosed herein; alternatively,they may be independent components and additives to a culture mediumdisclosed herein, for example, AAHKAU medium may be supplemented withcarbon dioxide obtained from combustion and thus have a differentcomposition than AAHKAU medium not supplemented with carbon dioxide oranother greenhouse or combustion gas. Thus, gases which can come out ofa mixture of flue gases can be used as components of AAHKAU medium.

Culture medium pH, salt content, osmolality, or temperature may beadjusted after addition of carbon dioxide or other combustion gases ortheir byproducts. In some embodiments, the culture medium, such asAAHKAU medium may be diluted to contain 10, 20, 30, 40, 50, 60, 70, 80,90, 100 or <100% of its 1× concentrations of one or more, or all, of itsingredients, for example, to compensate for effects of incorporating acombustion gas or combustion gas by product. Thus, an AAHKAU or relatedmedium may be used at a relative concentration of 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9 or <1× concentration. Preferably, the 1×concentrations of ingredients in AAHKAU medium or similar media are notreduced by more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%.

In other embodiments, AAHKAU medium may have a concentration of one ormore, or all, of its ingredients increased above the 1× concentrationsdisclosed herein, for example, AAHKAU medium may be used at aconcentration of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0× tocultivate microalgae. A decreased or increased concentration of AAHKAUmedium may be selected to minimize or otherwise compensate for additionof a greenhouse gas or greenhouse gas byproduct. A bioreactor comprisingHaematococcus sp. KAU-01 or a Haematococcus strain that has genomic DNAat least 99% identical to Haematococcus sp. KAU-01, an AAHKAU-likemedium and one or more ports and/or valves through which fresh mediummay be added to a culture and one or more ports and/or valves throughwhich spent medium and/or cellular debris may be removed from a culturewithout removing viable Haematococcus cells.

In some embodiments, the concentration of calcium nitrate Ca(NO)₃ inAAHKAU medium prevents cell lysis until the culture pH reaches 9.80.This results in higher biomass production by Haematococcus sp. KAU-01compared to other strains that lyse at a lower pH.

During addition of a gas mixture similar to coal power plant's flue gas,it was found that Haematococcus sp. KAU-01 grew in AAHKAU medium over awide range of pHs from 5.65 to 9.80 without rapturing of cells orforming cysts.

EXAMPLES

The following examples illustrate various aspects of the presentinvention. They are not to be construed to limit the claims in anymanner whatsoever.

Example 1 Isolation of Haematococcus sp. KAU-01

Isolation of Haematococcus sp. KAU-01. A sample was from Jeddah greenvalley where the water accumulates from several different mountainsusing a phytoplankton net with mesh size 10 micron. The samplinglocation was situated between 21° 36′71″N and 39° 43′16″E to 21° 32′75″Nand 39° 28′89″E; see FIGS. 8A-8D. Ten liters of water was filtered usingphytoplankton net and concentrated to 500 mL. Water samples werecollected from 10 locations. The concentrated sample was mixed with 10 Lof NOVA water (Saudi Arabian Nova Water—Health Water Bottling Co. Ltd.)enriched with F/2 medium stock solution (Aquacenter Inc., Leland, MI,USA; Guillard & Ryther, Studies of marine planktonic diatoms: I.Cyclotella nana husted, and Detonula confervacea (CLEVE) gran CANADIANJOURNAL OF MICROBIOLOGY, 1 Apr. 1962, 8(2); Guillard, Culture ofPhytoplankton for Feeding Marine Invertebrates, CULTURE OF MARINEINVERTEBRATE ANIMALS, 1975, pp 29-60; each incorporated by reference).

One mL of A and B solution was added with 10 liter of NOVA water to growmicroalgae which were grown in the transparent NOVA bottles. The culturewas grown under 12:12 hrs light:dark (L:D) cycles at 120 μmol photonsm⁻²s⁻¹ (μE m⁻² s⁻¹) and at temperature of 25° C. with moderate aerationfor 5 days. A one mL sample was taken into S-R chamber from each cultureand checked separately under light microscope.

A variety of different microalgae were observed including those ofHaematococcus sp., Scenedesmus sp., Pediastrum sp., Chlamydomonas sp.,Nostoc sp, and other diatoms found in wild culture samples. Afterchecking the cultures were kept without aeration for one day to formthin layer scum at the surface water.

When light was provided on the top of culture bottles it was observedthat Haematococcus formed thin layer scum at the surface of water if thewater was not mixed or agitated. A thin layer was observed at thesurface of the culture.

Tissue paper was applied to collect the microalgae from the surface. Thewet tissue paper was transferred to new culture bottle and the culturewas grown as described above. After 5 days, the culture turned green asobserved by naked eyes. Then, a 1 mL sample was taken into S-R chamberand microscopically observed. Many motile Haematococcus and fewChlamydomonas sp., microalgae cells were seen.

A single isolate of Haematococcus sp. was recovered using the methoddescribed and incorporated by reference to Affan, A., et al., Growthcharacteristics, biochemical composition and antioxidant activities ofbenthic diatom Grammatophora marina from Jeju coast, Korea. A LGAE,2006, 21, 141-48. Briefly, 1 mL of the mixed culture sample was dilutedwith 10 mL distilled water. Then, a 1 mL diluted sample was transferredto a Sedgwick Rafter (S-R) counting chamber, and single cell ofHaematococcus was picked up with mouth aspirated micropipette (MSM)which was made by heating the tip of a glass Pasteur pipette and thendrawing it to form a syringe type needle. The transparent PVC tube waspushed inside of the holder part of glass pasture pipette and finallythe joint between glass pasture pipette and PVC tube was made air tightwith vacuum latex tube. The length of the PVC tube was kept 75 cm whichwas convenient and comfortable to hold the tube into mouth and move theglass pasture pipette needle on the S-R chamber or into the multi-wellfor picking up the single cells. The needle of MSM was placed close tothe target a single cell which was observed under an inverted microscope(Olympus IX71) and gently aspirated to isolate the cell inside of theMSM.

Thereafter, each single cell was transferred to a multi-well plate forsubculture in culture medium which was prepared in distilled waterenriched with F/2 medium and autoclaved. The isolation process wascontinued until a mono specie was obtained. The mono species was thenstreaked onto an agar plate with 2% agar (w/v) and two mL/L of “A” and“B” stock solution F/2 and autoclaved distilled water. After two weeks,several green colonies were found in the agar plate.

A single colony from each plated was taken and put in plant tissuemulti-culture well plate which was filled with 7.5 mL F/2 enricheddistilled water culture medium. Thereafter, each well of multi well wasobserved under microscope to make sure that the isolated would be singlespecies. The agar plating and liquid culture were continued untilobtaining the single species of Haematococcus sp. Five mL of an aqueousculture of a single species (or isolate) of Haematococcus were pouredand mixed in flask which was filled with F/2 enriched agar medium attemperature of 35° C. when the agar was still semi-liquid. Thisprocedure resulted in the isolation of a mono species of Haematococcuswhich was designated Haematococcus sp. KAU-01.

Morphological and growth study in different reported media. The isolatedmicroalga Haematococcus sp. KAU-01 was examined under a light microscope(LM, Eclipse 80i; Nikon Co.). Images were obtained using a camera (DXM1200C; Nikon Co.). At first motile cells were observed discover thesimilarities or differences of their morphological characteristics withreported taxonomical characteristics. Then, life cycle study wasconducted and images were taken using same microscope as mentionedabove. After that, growth and biomass production study were conducted invarious culture media.

1^(st) step of culture in different culture media. The monostrainHaematococcus sp. KAU-01, was cultured in BG11 and other media describedin Table 1. The chemical composition and concentration of differentculture media (1×) used for growing Haematococcus sp. KAU-01 is shown inTable 1. It was cultured in BG11 incorporated by reference to Stanier,R. Y., et al., Purification and properties of unicellular blue-greenalgae (Order Chroococcales); see Media recipes, BACTERIOL. REV. 1971,1971. 35, 171-20; in MBG11 incorporated by reference to Allen, M. M &Stanier, R. Y., Growth and division of some unicellular blue-greenalgae. J. GEN. MICROBIOL. 1968, 51, 199-202; Watanabe, M. M., et al.,NIES-Collection List of Strains Sixth Edition 2000 Microalgae andProtozoa. MICROBIAL CULTURE COLLECTIONS, NATIONAL INSTITUTE FORENVIRONMENTAL STUDIES, Tsukuba, 159 pp; in OHM incorporated by referenceto Fabregas, J., et al., Two-stage cultures for the production ofastaxanthin from Haematococcus pluvialis. JOURNAL OF BIOTECHNOLOGY.2001, 89: 65-71), in Basal medium incorporated by reference to Hata, N,et al., Production of astaxanthin by Haematococcus pluvialis in asequential heterotrophic-photoautotrophic culture. JOURNAL OF APPLIEDPHYCOLOGY 2001, 13: 395-402; in RM (Rudic's medium) incorporated byreference to Rudic, V. & Dudnicenco, T., Process for cultivation ofgreen alga Haematococcus pluvialis (Flotow). 2000, MD Patent Nr. a 20000154), in HK (Hong-Kong) medium (Kamonpan Kaewpintong, K., et al.Photoautotrophic high-density cultivation of vegetative cells ofHaematococcus pluvialis in airlft bioreactor BIORESOURCE TECHNOLOGY,2007, 98: 288-295; and in MCM incorporated by reference to Borowitzka,et al., Culture of the astaxanthin-producing green alga Haematococcuspluvialis 1. Effects of nutrients on growth and cell type, JOURNAL OFAPPLIED PHYCOLOGY, 1991, 3, 295-304.

TABLE 1 Nutrient composition and concentration (mg/L) of differentculture media for Haematococcus sp. KAU-01. BG11 MBG11 OHM Basal RM HKMCM Concentrations Ingredient mg/L mg/L mg/L mg/L mg/L mg/L mg/L HNO₃NaNO₃ 1500 1500 300 KNO₃ 410 300 200 K₂HPO₄ 40 320 80 20 KH₂PO₄ 150 20Na₂HPO₄ 30 30 NaH₂PO₄ 35.5 H₃PO₄ Ca(NO₃)₂•4H₂O 100 CaCl₂•2H₂O 36 36 11058.5 73 80 MgSO₄•7H₂O 75 200 246 40 10 24.6 100 Mg(NO₃)₂•6H₂O Na₂CO₃ 20100 NaCl 20 Urea β-Na₂glycero 50 phosphate Citric acid 6 6(NH₄)₅[Fe(C₆H₄O₇)]₂ 6 6 EDTA-Na₂ 1 1 2.71 6.7 EDTA 7.5 0.0198 VitaminB12 0.0015 0.0001 0.004 Biotin 0.025 0.0001 Thiamine HCl 0.01 Thiamine0.0175 H₃BO₃ 286 286 0.3 0.003 61 MnCl₂•4H₂O 1.81 1.81 0.98 0.108 4.1MnSO₄•H₂O 1.5 0.001 ZnSO₄•7H₂O 0.22 0.22 0.066 0.1 0.014 4.1Na₂MoO₄•2H₂O 0.39 0.39 12.0 0.0075 0.001 (NH₄)₆Mo₇O₂₄•4H₂O 0.3 38.0CuSO₄•5H₂O 0.08 0.08 0.012 0.08 0.012 6.0 Co(NO₃)₂•6H₂O 0.05 0.05 0.26FeCl₃•6H₂O 5.888 0.0244 FeSO₄•7H₂O 8.3 CoCl₂•6H₂O 0.11 0.12 0.0005 5.1Trisaminomethane 500 Fe(III)citrateH₂O 2.67 Cr₂O₃ 0.075 SeO₂ 0.005

Media were made with the respective amounts of chemical ingredientsdescribed above, diluted in distilled water and then autoclaved for 15mins. A 1.5 liter medium was made for each of culture media recipes.Each culture was grown in one liter Erlenmeyer Flask with 500 mL of eachculture medium at temperature of 25° C. under fluorescent lights (120 μEm⁻² s⁻¹) on a 12:12 h L:D photo cycle with moderate aeration for 16days. All cultures were conducted in triplicate (n=3).

Determination of growth and biomass production. Growth of Haematococcussp. KAU-01 was determined in two ways, one was direct cell counting andanother weighing dry biomass. Samples were collected from each flaskevery other day. For dry biomass estimation, a 20-mL sample wascollected from each culture flask, filtered through preweighed GF/FWhatman filter paper. A preweighed filter paper that was soaked indistilled water and dried at the same time was used as a blank. Thebiomass filter paper was kept at 55° C. in an oven, dried and weighed,and the dry weight biomass was calculated as gL⁻¹. For cell counting, a5-mL sample was collected from each culture flask and fixed with 2% ofLugol's iodine solution. The fixed sample was diluted and the cells werecounted using a S-R counting chamber under an inverted microscope. Drybiomass and cell counting values were used for plotting as a growthcurve.

The specific growth rate (p), defined as the increase in cell density ordried biomass per unit time was calculated and formulated as follows:

$\mu = \frac{\ln\mspace{11mu}\left( {X_{1}\text{/}X_{0}} \right)}{t_{1} - t_{0}}$

Where X₀ and X₁ are cell density/dried biomass at the beginning (t₀) andend (t₁) of a selected time interval between inoculation and maximumcell density dried biomass, respectively. For determining the growthcurve of each sample, replicates were counted and the mean value wasused.

Preparation of Haematococcus sp. KAU-01-culture medium. The conditionand biomass production of Haematococcus sp. KAU was dependent on themedium they were cultured in. This may be related with differentchemicals with different concentrations in above mentioned differentcultures media. Some cells lysed during culture in different media andthe biomass production was gradually decreasing. Based on theseobservations, the inventors sought to increase biomass production byproviding media and conditions that would keep cells in a motiledivision state and that would subsequently increase biomass productionin a cyst state which is important for industrial scale production ofbiomass.

The inventors observed that there were more lysed cells in culture mediawhich were prepared without calcium, that cells were weak and platerather than dark green when a culture medium has a low concentration ofmagnesium, and that a high concentration of Ca(NO₃)₂ arrested culturegrowth and caused milky turbidity at a pH above 8.50.

Another coincident negative effect microscopically observed in thesecultures was that normal, motile swimming mode was disrupted resultingin slow moving or stationary cells when cellular debris aggregated tothe flagella and periphery of the transparent cell walls.

Consequently, the inventors developed new culture media that avoided orameliorated these problems, for example, by gradually adding ortitrating Ca and Mg salts such as Ca(NO₃)₂ and Mg(NO₃)₂.6H₂O to select aconcentration of Ca and Mg that did not negatively impact cell growthand biomass production. The concentration of Ca and Mg were 4.83 and6.46 milligram per liter in the AAHKAU medium formulation showed thebest growth of Haematococcus sp., KAU-01. These may be slightly higherwhen water containing Ca and Mg is used to produce the medium.Preferably, the above concentrations of Ca and Mg do not vary by morethan ±1, 2, 3, 4, 5, 10, 15 or 20% in modified forms of AAHKAU mediumand preferably, any added Ca and Mg is added as a nitrate salt.

Media stock solutions containing major nutrients such as major nutrientsNaNO₃, KNO₃, Ca(NO₃)₂, Mg(NO₃)₂.6H₂O, K₂SO₄, K₂HPO₄, KH₂PO₄, MgSO₄·7H₂O,urea, HNO₃, and H₃PO₄; and as micro nutrients FeCl₃·6H₂O, H₃BO₃,Co(NO₃)₂·6H₂O, K₂Cr₂O₇, CuSO₄·5H₂O, MnSO₄·H₂O, ZnSO₄·6H₂O, Na₂-ETDA, andHCl were formulated as disclosed below. For the vitamin solution,thiamine, +D-biotin, and cyanocobalamin were diluted in autoclaveddistilled water and during culture 100 μl/L the vitamin stock solutionwas added in one liter of culture medium.

2^(nd) step of culture to select best culture media. Three stocksolutions were prepared, major nutrients (“A”), micronutrients (“B”) andvitamins (“C”). Distilled water, filtered natural seawater andautoclaved distilled water was used to make stock solution of “A”, “B”,and “C”, respectively. Then, five concentrations of “A”, fiveconcentrations of “B” and a one concentration of “C” stock solutionswere added to freshwater to culture of Haematococcus sp KAU.

The chemical concentrations of 1000× stock solution “A” were NaNO₃(375.00 g), KNO₃ (75.00), Ca(NO₃)₂ (25.00 g), Mg(NO₃)₂·6H₂O (55.00 g),K₂HPO₄ (45.00 g), KH₂PO₄ (40.00 g), K₂SO₄ (10.00 g), MgSO₄·7H₂O (23.75g), urea (1.75 g), HNO₃ (65.00 ml/L) and H₃PO₄ (15 ml/L) which werediluted in autoclaved distilled water and the final volume was made 1 L.

For 10,000× stock solution “B”, FeCl₃·6H₂O (35.00 g), H₃BO₃ (10.00 g),Co(NO₃)₂·6H₂O (2.50 g), K₂Cr₂O₇ (1.00 g), CuSO₄·5H₂O (1.00 g), MnSO₄·H₂O(2.50 g) ZnSO₄·6H₂O (7.50 g), (NH₄)₆Mo₇O₂₄·4H₂O (2.50 g), Na₂-ETDA(10.00 g) and HCl (10.00 ml) were diluted in filtered natural seawaterand finally the volume was made 1 L.

For 10,000× stock solution “C”, thiamine (10.00 g), +D-biotin (0.03 g)and cyanocobalamin (0.001 g) were diluted in autoclaved distilled waterand finally the volume was made 1 L.

Thereafter, five concentrations of “A”, “B” and one concentration of “C”were diluted for making culture medium to test growth of HaematococcusKAU sp.

The diluted concentration of “A” stock solution was 0.5, 0.75, 1.00,1.25 and 1.50 ml/L to prepare ‘A1’, ‘A2’, ‘A3’, ‘A4’ and ‘A5’ culturemedium, respectively.

Similarly, the diluted concentration of “B” stock solution was 50.00,75.00, 100.00, 125.00 and 150.00 μl/L in ‘A1’, ‘A2’, ‘A3’, ‘A4’ and ‘A5’culture medium, respectively. Stock solution C was added in an amount of100.00 μl/L to each of ‘A1’, ‘A2’, ‘A3’, ‘A4’ and ‘A5’ culture medium,respectively. The chemical concentration given in Table 5, is the final1× concentration for culture medium and the concentration unit is mg/Lfor salts and μl/L for acids. In Tables 2, 3 and 5, the concentrationsof chemicals are given in mg/L and μl/L.

Tables 2, 3 and 4 show ingredients in each major (A1-A5) and micro stocksolution (B1-B5) and square design among major and micronutrients(X1-X5). such as A1, A2, A3, A4 and A5, and five concentrations ofmicronutrients stock solutions such as B1, B2, B3, B4 and B5, and groups(X1-X5) for square design among major nutrients and micronutrients.

Results of cultivating Haematococcus sp. KAU-01 in the mediacombinations described by Table 4 are shown by FIGS. 13A-13E. FIG. 13Fshows the superior dry biomass produced by culturing Haematococcus sp.KAU-01 in combination A3B3 (AAHKAU) medium in comparison to severalconventional culture media.

TABLE 2 Major Nutrients Group A B C D E A1 A2 A3 A4 A5 Chemicals namemg/L mg/L mg/L mg/L mg/L NaNO₃ 187.50 281.25 375.00 468.75 562.5 KNO₃37.50 56.25 75.00 93.75 112.5 Ca(NO₃)₂ 12.50 18.75 25.00 31.25 37.50Mg(NO₃)₂•6H₂O 27.50 41.25 55.00 68.75 82.50 K₂HPO₄ 22.50 33.75 45.0056.25 67.5 KH₂PO₄ 20.00 30.00 40.00 50.00 60.00 K₂SO₄ 5.00 7.50 10.0012.50 15.00 MgSO₄•7H₂O 11.88 17.81 23.75 26.69 35.63 Urea 0.88 1.31 1.752.19 2.63 HNO₃ 32.50 μl 48.75 μl 65.00 μl 81.25 μl 97.50 μl H₃PO₄  7.50μl 11.25 μl 15.00 μl 18.75 μl 22.50 μl

TABLE 3 Micronutrients Group B1 B2 B3 B4 B5 Chemicals name mg/L mg/Lmg/L mg/L mg/L FeCl₃•6H₂O 1.75 2.63 3.50 4.38 5.25 H₃BO₃ 0.50 0.75 1.001.25 1.50 Co(NO₃)₂•6H₂O 0.13 0.19 0.25 0.31 0.38 K₂Cr₂O₇ 0.05 0.08 0.100.13 0.15 CuSO₄•5H₂O 0.05 0.08 0.10 0.13 0.15 MnSO₄•H₂O 0.13 0.19 0.250.31 0.38 ZnSO₄•6H2O 0.38 0.56 0.75 0.94 1.13 (NH₄)₆Mo₇O₂₄4H₂O 0.13 0.190.25 0.31 0.38 Na₂-ETDA 0.50 0.75 1.00 1.25 1.50 HCl 0.500 μl 0.750 μl0.100 μl 0.1250 μl 0.150 μl

TABLE 4 Design combinations X1 X2 X3 X4 X5 A1 B1 A2B1 A3B1 A4B1 A5B1 A1B2 A2B2 A3B2 A4B2 A5B2 A1 B3 A2B3 A3B3 A4B3 A5B3 A1 B4 A2B4 A3B4 A4B4A5B4 A1 B5 A2B5 A3B5 A4B5 A5B5

An advantageous formulation described by Table 5 (“A3B3” formulationdescribed by the combination of Tables 2 and 3) was designatedAffan-Adnan Haematococcus King Abdulaziz University or “AAHKAU” culturemedium. The concentration of elemental Ca and Mg were 4.83 and 6.46milligram per liter in the AAHKAU medium formulation. These may beslightly higher when water containing Ca and Mg is used to produce themedium. “Elemental” Ca or Mg refers to the molecular mass of the Ca orMg component of a compound or salt such as Ca(NO₃)₂.

To avoid or minimize turbidity and enhance growth of Haematococcus theinventors found that it was important to regulate the concentration ofCa (elemental) and Mg (elemental) from all ingredients in AAHKAU mediumor its derivatives between 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6.0,6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 mg/L for elemental Ca and between 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, and 11 mg/L for elemental Mg. Preferably the minimum and maximumconcentrations of elemental Ca range from 2.41 to 7.24 mg/L (or anyintermediate value within this range) and those of elemental Mg rangefrom 3.23 and 9.69 mg/L (or any intermediate value within this range).Typically, the concentration of elemental Ca is regulated to avoidturbidity.

Alternative formulations of AAHKAU medium may be used to cultureHaematococcus include modified AAHKAU medium produced by othercombinations of A1-A5×B1-B5 (see Table 4, for example) or produced byvarying the concentration of one or more ingredients in formulations A3or B3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or50%. Affan-Adnan-Haematococcus-King Abdulaziz University (AAHKAU).

TABLE 5(A) Chemical composition and 1X (mg/L and μml/L) concentrationsof Haematococcus sp KAU culture medium AAHKAU for the obtaining rapidgrowth and biomass production. 1X AAHKAU Medium A-solution Conc.B-Solution Conc. C-Solution Vitamins Chemicals mg/L Chemicals mg/LChemicals mg/L NaNO₃ 375.00 FeCl₃•6H₂O 3.50 Thiamine 1.00 Ca(NO₃)₂ 25.00H₃BO₃ 1.00 +D-biotin 0.003 Mg(NO₃)₂•6H₂O 55.00 Co(NO₃)₂•6H₂O 0.25Cyanocobalamin 0.0001 K₂HPO₄ 45.00 K₂Cr₂O₇ 0.10 KH₂PO₄ 40.00 CuSO₄•5H₂O0.10 KNO₃ 75.00 MnSO₄•H₂O 0.25 K₂SO₄ 10.00 ZnSO₄•6H2O 0.75 MgSO₄•7H₂O23.75 (NH₄)₆Mo₇O₂₄•4H₂O 0.25 Urea 1.75 Na₂-ETDA 1.00 HNO₃ 65.00 μl HC11.00 μl H₃PO₄ 15.00 μl

The pH is typically adjusted to 7.5 by adding Na₂CO₃.

TABLE 5(B.) The chemicals name and concentrations for a 1000X stocksolution (g/L or ml/L) of A-solution and 10000X stocks of B- andC-solutions for AAHKAU medium for Haematococcus sp., KAU-01. StockSolutions A-solution B-Solution C-Solution (1000×) Conc. (10,000×) Conc.(10,000×) Vitamins Chemicals g/L Chemicals g/L Chemicals g/L NaNO₃375.00 FeCl₃•6H₂O 35.00 Thiamine 10.00 Ca(NO₃)₂ 25.00 H₃BO₃ 10.00+D-biotin 0.03 Mg(NO₃)₂•6H₂O 55.00 Co(NO₃)₂•6H₂O 2.50 Cyanocobalamin0.001 K₂HPO₄ 45.00 K₂Cr₂O₇ 1.00 KH₂PO₄ 40.00 CuSO₄•5H₂O 1.00 KNO₃ 75.00MnSO₄•H₂O 2.50 K₂SO₄ 10.00 ZnSO₄•6H₂O, 7.50 MgSO₄•7H₂O 23.75(NH₄)₆Mo₇O₂₄•4H₂O 2.50 Urea 1.75 Na₂-ETDA 10.00 HNO₃ 65 ml HCl 10.00ml/L H₃PO₄   15 ml/L

This advantageous formulation was designated Affan-Adnan HaematococcusKing Abdulaziz University or “AAHKAU” culture medium. In someembodiments, an AAHKAU-like medium may be produced by varying theconcentrations of one or more of the above-named ingredients by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50%. Moreconcentrated stock solutions of the A-, B- and C-solutions in Table 5may be formulated by increasing the amounts of chemical ingredients withrespect to water content. For example, a 1000× concentrated solution maybe produced by replacing the units in Table 5 “mg/L” for salts andvitamins with g/L and replacing “μl/L” for acids with “mL/L”. A 1×solution can then be produced by diluting 1 ml of 1000× concentratedstock solution-A and 0.1 ml of each of 10,000× stock solutions B and Cup to 1 L with water thus producing a 1× medium for growingHaematococcus sp. KAU-01.

3^(rd) step of culture. After formulation of the medium, Haematococcussp. KAU-01 was grown in AAHKAU medium and other reported media (OHM, RM,Basal and HK media) to compare the growth performance. For growth studyin AAHKAU medium, two types of water were used, one was distilled waterwhich was name as AAHKAU-1 and another was tap water from the municipalwater supply which was named as AAHKAU-2. Both of AAHKAU-1 and AAHKAU-2were kept 4 hours after adding of stock solutions of AAHKAU culturemedium at low pH (2.88), since stock solution-A and B were highly acidic(pH˜0.18). The pH was then adjusted to 7.00 by adding Na₂CO₃. Theculture was done in two liter Erlenmeyer flasks containing 1000 mL ofeach culture medium. The culture comparisons were conducted at samelight intensity and temperature with L:D cycle of 12:12 hr. Gentleaeration was provided continuously provided to agitate the culture.

Microscopic observations. Morphological characteristics of the isolatedmicroalga were investigated under inverted light microscope (LM) andphotographed. LM based analysis was done from the mono species isolatedHaematococcus sp. KAU-01 microalga.

Vegetative motile cell with two flagella, semitransparent trilaminarsheath between cytoplasm, cytoplasmic strands that attaches the maincell body to the theca or the outer cell wall and pyrenoids wereobserved under inverted light microscope (FIG. 9 ). The shapes ofbiflagellate vegetative cells were ovoid to ellipsoidal averaging27.7±3.3 μm in length and 22.0±4.2 μm in diameter (FIG. 10A).

During the laboratory culture, the flagellated cells were changed toimmotile intermediate cells which were palmelloid with some astaxanthinaccumulation; see FIGS. 10A and 10B. Within 2-3 days the cells turnedinto red cysts cells or aplanospores; FIG. 10C. An average diameter ofboth immotile with central red and green toward periphery or redspherical palmelloid cells were 35.6±8.2 μm. The average diameter of therelatively large red cyst cells diameter was above 65 μm; FIGS. 10B and10C.

The red cyst started asexual reproduction after 2 days of inoculationinto new culture medium (FIG. 10D and FIG. 10E) then the daughter cellscame out by breaking cell wall (FIG. 10F). Cells of the palmelloid stagereproduced asexually by cell division and formed 4 to 64 zoospores (FIG.10D and FIG. 10E).

In other culture media, lysed cells were seen and sometimes wholecultured cells or some of culture was found to be lysed without formingcyst. Lysis could be stopped by gradually increasing the concentrationof Ca(NO₃)₂ up to 25.00 mg/L (4.83 mg/L elemental Ca) which is theamount in 1×AAHKAU medium as this concentration was considered as theprimary concentration for making the AAHKAU Haematococcus sp. KAU-01culture medium. The fate of cells cultured without addition of Ca(NO₃)₂is shown in FIGS. 11A-11D.

In some embodiments or variations of this novel medium, an elementalcalcium concentration may be selected as an amount sufficient to preventor sufficient reduce lysis to 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 or90% of a control lysis value of a culture having no or less than 1, 2,3, 4, 5, or 6 mg/L elemental calcium. Preferably the content range ofelemental Ca is 2.41 to 7.24 and the content range of elemental Mg is3.23 to 9.69 mg/L in AAHKAU medium or a variation thereof.

Growth and biomass production in different culture media in 1^(st) stepof culture. The dry biomass production (g/L⁻¹) differed depending on theselected culture media. Moreover, peak growth in view of biomassproduction occurred at different times or different days depending onthe culture medium selection. In BG11 culture medium, the highestbiomass production was 1.18 gL⁻¹ on 10^(th) day of culture. Similarly,in MCM, MBG11 or OHM culture medium the highest biomass production,respectively, 1.22, 1.22 and 1.16 gL⁻¹ on 12^(th) day of culture.Biomass production in Basal, HK and RM culture media were 1.18, 1.29 and1.35 gL⁻¹ on 14^(th) day of culture (FIG. 12A). However, the highestbiomass production was in RM culture medium, followed by HK and Basalmedia (FIG. 12A).

The result of cell abundance Haematococcus sp. KAU-01 also showedsimilar growth pattern as it was found in dry biomass estimation. InBG11 culture medium the highest cell abundance was 8.19×10⁵ cells L⁻¹ on10^(th) day of culture. Similarly, in MBG11, OHM and MCM culture media,the maximum cell abundance respectively was 8.19×10⁵, 8.27×10⁵ and8.40×10⁵ cells L⁻¹ on 12^(th) day of culture. However, the highest cellabundance among the culture was 9.67×10⁵ cells L⁻¹ in RM culture medium,followed by HK (9.20×10⁵) and basal (8.34×10⁵) culture media on 14^(th)day of culture (FIG. 12B).

Growth of Haematococcus sp. KAU-01 in AAHKAU culture medium in the2^(nd) step of culture. Haematococcus sp. KAU grew well in all squarecombinations of ‘A and B stock solutions mixed culture media. The higherbiomass production was in major nutrient C group (media combinationsA3B1; A3B2; A3B3; A3B4; A3B5) than those of A, B, D and E groups. In Cgroup, the biomass production was 1.86 gL⁻¹ in A3B3, followed by A3B4and A3B5 (FIGS. 13A, 13B, 13C, 13D and 13E). Therefore, AAHKAU (A3B3)culture medium recipe was considered to be the most advantageous mediumfor growth of Haematococcus sp. KAU-01 and designated AAHKAU medium.

For the final step of culture in selected AAHKAU and reported media aHaematococcus sp. KAU-01 growth experiment was conducted to compare thegrowth and biomass production with other conventional media.Haematococcus sp. KAU-01 was grown in OHM, Basal, RM, HK and AAHKAU(A3B3) culture media. The biomass production was highest 2.20 gL⁻¹ inAAHKAU medium, followed by RM (1.37 gL⁻¹) and HK (1.31 gL⁻¹),respectively (FIG. 14A). Maximum specific growth (μ_(max) d⁻¹) rate was0.161 and 0.163 d⁻¹ on 12^(th) day of culture in basal and OHM culturemedia.

The highest the μ_(max) d⁻¹ was 0.179 d⁻¹ on 14^(th) day of culture inAAHKAU-1, followed by 0.150 and 0.146 d⁻¹ in RM and HK media,respectively on same day of culture. In case of AAHKAU-2, the biomassproduction, cell abundance and μ_(max) d⁻¹ were 1.96 gL⁻¹, 14.13×10⁵cells L⁻¹ and 0.174 d⁻¹, respectively (FIG. 14B).

Morphological features of Haematococcus sp. KAU-01 are illustrated inFIG. 9 . The motile cell having several pyrenoids, two flagella andcytoplasmic strands connecting with main cell body and outer cell wallwere observed under inverted light microscope. The motile flagellatedcell typically exhibits a voluminous, transparent and gelatinous lookingcell wall which is the characteristic of Volvocalean motile cells.Haematococcus sp. are characterized by ovoid, ellipsoid or sphericalcells and 34-37.5 μm in diameter (up to 5 μm broad and 63 μm long). Thecell wall is widely separated from the protoplast without a papilla,protoplast with a beak-like apex not reaching up to the wall, plasmastrands branched and connected with cell wall, chloroplast with thickmembrane having 6-8 (up to 15) scattered pyrenoids. These microscopiccharacteristics are similar to motile cells of Haematococcus sp. KAU-01.

During the laboratory culture, the flagellated cells converted toimmotile intermediate cells (palmelloid with some astaxanthinaccumulation). Those cells were picked up following MSM technique andkept into multiwall tissue culture plate with distilled water under samelight condition as culture. After 3 days all intermediate cells turninto deep red cyst. The red cysts again inoculated into fresh media andobserved after certain interval for changing to different stage of lifecycle. There were 4 to 64 daughter cells were observed in the red cystand after few hours the daughter cells ruptured the cell wall of thecyst and motile cells came out and start to multiply. A similarobservation was made by Hagen et al. (2001) who indicated that thedevelopmental cycle of Haematococcus pluvialis, flagellates are formedby germination of resting cells after getting favorable environmentalcondition. Consequently, the inventors place their isolate within thegenus Haematococcus sp. based on light microscopic study of motile cellsand life cycle of this alga. This new isolate and species within thisgenus was designated Haematococcus sp. KAU-01.

During culture of Haematococcus sp. KAU-01, the broken or lysed cellswere observed in the culture medium prepared without or with lowconcentrations of calcium salts. The cell wall of Haematococcus sp.KAU-01 was not broken or ruptured when Ca(NO)₃ concentrations rangedfrom 12.50 to 37.50 mgL⁻¹ in culture medium where 25.00 mgL⁻¹ of Ca(NO)₃gave the best result for growth. A higher concentration was found tostop the movement of flagellated motile cells and inducing to form cyst.The inventors observed that Haematococcus sp. KAU-01 grew between pH5.65 and 9.80 without significant lysis or rupture of cells or formationof cysts. They also found that a suitable concentration of calciumprotected Haematococcus from lysis at least until the pH of the culturemedium exceeds 9.8. This inhibition of cell lysis enhanced biomassyield. Additionally, calcium nitrate salts were used rather than calciumchloride or calcium phosphate. During photosynthesis chloride ions playa role in balancing of potassium and in water splitting however, nitrateplays an important role as a major plant nutrient. Calcium phosphatemakes the culture medium milky turbid when adjusted to pH 7.5. Thenitrate supplied by calcium nitrate also plays a role in biosynthesis ofamino acids and for total protein and carbohydrate production of cells.

Growth and biomass production in different culture media. The growthrate and biomass production of Haematococcus sp were influenced byseveral physical and chemical factors. Chemical factors includeselection of proper combinations of macro and micronutrients andphysical factors include culture temperature and illumination. Theinventors found that Haematococcus sp. KAU-01 showed similar growth asH. pluvialis when Haematococcus sp. KAU-01 was cultured under at lightintensity of 120 μE m⁻² s⁻¹ on a 12:12 L:D photo cycle. However,surprising results were obtained when Haematococcus sp. KAU-01 was grownin AAHKAU medium and other reported media at 100 μE m⁻² s⁻¹ on a 12:12 hL:D photo cycle. The cell abundance and biomass production were higherin light intensity of 100 μE m⁻² s⁻¹ than that of 120 μE m⁻² s⁻¹ undersame L:D photo cycle intensity in all culture media.

Among the all culture, the highest cell abundance, μmaxd⁻¹ and biomassproduction were in AAHKAU culture medium. RM, BG11 and Basal media aremore often used for growing H. pluvialis. Among those media, RM andbasal media showed the higher cell abundance which was 9.50×10⁵ and8.85×10⁵ cells mL-1, and the maximum specific growth rate was 0.195 and0.177 d-1, respectively. The remarkable growth, cell abundance andbiomass production achieved in AAHKAU medium exceeded that of any otherreported culture media. While not being bound to any theory orexplanation, the inventors believe that the Ca²⁺, Mg²⁺, and NO³⁻ contentof AAHKAU medium in combination with large amount of iron may protectagainst cell wall lysis, increase chlorophyll content and proteinsynthesis and ultimately enhance growth and biomass production.

To test the effects of culturing Haematococcus in tap water acomparative AAHKAU-2 culture was performed using tap water to observethe growth and biomass production by eradication of contaminant speciesby adding of acidic stock solution of AAHKAU. In AAHKAU-2 culture,Haematococcus sp. KAU-01 grew well in comparison with reported media andthere were no more contaminant species found. In tap water somemicroalgae (Scenedesmus sp. Chlamydomonas sp., etc.) was found to growafter enriched with F/2 culture medium nutrients during trial errorstudy of growth of Haematococcus sp. KAU-01. It was also observed thatgrowth of Haematococcus sp. KAU was stopped in presence of contaminatingmicroalgae, for example, Haematococcus sp. KAU-01 was lost from aculture containing Scenedesmus sp. KAU-01. Biomass production was lowerin AAHKAU-2 (tap water) than that the biomass production of AAHKAU-1.

In Jeddah, tap water for domestic use is supplied from desalinationplants for domestic use. The desalinated water contains 100-1000 mgL⁻¹of total dissolved solids, and the concentration of calcium is almost200 mgL⁻¹ of water; Hussein, M. & Magram, S. F., Domestic Water Qualityin Jeddah, Saudi Arabia. JKAU: ENG. SCI. 2012, 23, 207-223. Highconcentrations of Ca²⁺ might have played role to slow growth ofHaematococcus sp. KAU-01 as discussed above.

This Example shows for the first time the morphological characteristicsand preparation of optimum culture medium for isolated Haematococcus sp.KAU-01 in KSA and the GCC. The new strain grew in all the reportedculture media. Additionally, it showed massive growth in newly optimizedculture medium (AAHKAU) in batch culture. Ca salt concentration inAAHKAU medium was confirmed to prevent or resist cell wall lysis. AAHKAUmedium stock solutions also conferred the ability to eradicate microbialcontaminants found in water that is not autoclaved such as tap water.Consequently, autoclaving of water or culture medium is not neededbefore inoculation of Haematococcus sp. for their culture and thisprovides a way to perform large scale outdoor culture for industrialscale biomass using AAHKAU culture medium, particularly in hot tropicalcountries like the Kingdom of Saudi Arabia and GCC countries usingHaematococcus sp. KAU-01 which was locally isolated from a hot tropicalclimate.

Example 2

Culture of Haematococcus sp. KAU-01 with Normal Aeration and/or inPresence of Combustion Gases.

As shown herein the inventors have developed a culture medium andmethods useful for convenient and economical large scale biomassproduction from microalgae that mitigates or eliminates atmosphericpollution caused by flue gases emissions from fossil fuel power plantswhile enhancing microalgae growth and biomass production.

Materials and methods. Three stock solutions, “A” for major nutrients,“B” for micronutrients and “C” for vitamins were prepared as describedabove. To produce a 1× AAHKAU culture medium as described in Table 5approximately 1.0, 1.0 and 1.0 ml of stock solution of ‘A’, ‘B’ and ‘C’were respectively diluted into 1 L of water. Two types of water used toprepare the culture medium, one was distilled water and another one wasmunicipal water. After dilution of stock solutions, the water pH waschecked which was about 3.0 and Na₂CO₃ was added to adjust the pH to pH7.00 prior to inoculating the 1× medium with Haematococcus sp. KAU-01for growth and biomass production. The study was carried out until allthe motile cells turn to inactive cysts.

Three groups of culture experiments A, B and C were designed to identifythe best culture conditions for with Haematococcus sp. KAU-01.

The cultures in Group A (A1, A2 and A3) were continuously aerated withnormal air to agitate the cultures, those in Group B (B1, B2 and B3)were given 15% CO₂ gas periodically after day 4 of culture along withcontinuous normal aeration, and those in Group C (C1, C2 and C3) weregiven mixture gases (15% CO₂+5% CO+1% NO+1% NO₂) periodically after thefourth day of culture along with continuous normal aeration.

The A1 culture was done with adding of initial concentrations of stocksolutions of AAHKAU medium (solution A, B and C of AAHKAU medium at 3.5,1.0 and 1.0 ml/L) to produce 1× medium.

Initial nutrients concentration of A2 medium was same as regards to theinitial nutrients concentration of A1, and after four days of culture acontinuous nutrients of AAHKAU medium was provided to the A2 culture.For that, initial concentrations of stock solutions of AAHAKU medium wasdiluted 2.5 times in distilled water. Thereafter, the diluted stocksolutions were added continuously to maintain constant pH of 7.5, sincestock solution of AAHAKU medium is highly acidic. The diluted AAHKAUmedium was added with a pH controller and dosing pump (BL7916-2 pHController with Pump, Hanna, Instrument Co.)

The A3 culture was same as regards to A1 culture except exchange of 20%ongoing culture medium with fresh initial concentration of AAHKAU mediumconcentration after four days of culture. The exchange of culture mediumwas done on every third day.

In B1 culture, CO₂ gas was provided periodically after four days ofculture to adjust the pH 7.5. CO₂ gas was provided to the culture duringthe light period to adjust pH of 7.5. The culture pH was checked hourlyand CO₂ gas was provided to the culture.

The B2 culture was the same as the B1 culture except for addition of 2.5times dilute, initial concentration of AAHKAU medium stock solutions toadjust pH of 7.5 once in a day after four days of culture while rest ofthe time the pH was adjusted 7.5 with periodically providing of CO₂ gasto the culture. CO₂ gas was provided following the same way as describedin B1 culture.

The B3 culture was same as regards to B1 culture except exchange of 20%ongoing culture medium with fresh initial concentration of AAHKAUmedium. The exchange of 20% ongoing culture medium of B3 was same asregards to A3 culture. And CO₂ gas was provided as described for the B1culture.

In the C1 culture, the mixture gages (CO₂ 15%+CO 5%+NO 1%+NO₂ 1%+N₂balance) was provided periodically after four days of culture to adjustthe pH of 7.5. The mixture gases were provided following the same way asdescribed for the B1 culture.

The C2 culture was same as the C1 culture except for addition of 2.5times diluted stock solution of AAHKAU medium once in a day to adjust pH7.5 after four days while the rest of the time pH was adjusted 7.5 withmixture gases. Periodically providing of mixture gases was same asdescribed for the C1 culture.

The C3 culture was same as regards to C1 except exchange of 20% ongoingculture medium with fresh initial concentration of AAHKAU medium. Theexchange of 20% medium was same as regards to A3 culture. Periodicalproviding of mixture gases was same as described for the C1 culture.

Exchange of culture medium in A3, B3 and C3 was done by discharging of20% old culture medium through filtration and replacement of newlyprepared culture medium (initial concentrations of nutrients). Forexchanging, a 5.00 micron-mesh size filter bag (Zhengzhou MiningMachinery, Co. Ltd, China) was cut and sacks were made. Sacks were 15 cmlong and 10 cm wide with a small opening where one side of clearflexible 4 mm air tube of 1.5 meter length was pushed inside of sacksand fixed with Gorilla super glue and cellophane. Then, one side of veinsaline tube was pushed inside of the flexible tube and fixed tightlywith cellophane and another part a regulator was kept to control the outflow of culture medium. Thereafter, the culture medium was allowed todischarge by gravitation force and flow speed was controlled by theregulator. The alga that attached to the sack's surface was removed byreverse forced air flow.

Culture medium exchange was stopped when 90% motile cells turned intocysts. The culture was grown in transparent 12 L NOVA bottles (HealthWater Bottling Company, the Kingdom of Saudi Arabia, hypertext transferprotocol://worldwideweb.novawater.com/en/index.php).

The upper parts of the bottles were cut to form a cylindricalphotobioreactor. Each bottle was filled up with 10 liters AAHKAU culturemedium. The culture was grown at temperature of 25° C. under fluorescentlights (180 μE m⁻² s⁻¹) on a 14:10 h L:D light dark photo cycle for 26days. All cultures were conducted in triplicate (n=3).

Determination of growth and biomass production. The growth ofHaematococcus sp. KAU-01 was determined in two ways. One was direct cellcounting and the other weighing dry biomass. Samples were collected fromeach flask every other day.

For dry biomass estimation, a 20 mL sample was collected from eachculture flask, filtered through preweighed GF/F Whatman filter paper. Apreweighed filter paper that was soaked in distilled water and dried atthe same time was used as a blank. The biomass filter paper was kept at55° C. in an oven, dried and weighed, and the dry weight biomass wascalculated as g/L.

For determination of particulate materials, 250 mL discharge water wasfiltered and dried using filter paper following the same procedure usedfor biomass determination and expressed as mg/L.

For cell counting, a 5 mL sample was collected from each culture flaskand fixed with 2% of Lugol's iodine solution. The fixed sample wasdilute and the cells were counted using a S-R counting chamber under aninverted microscope.

A growth curve was plotted using the dry biomass and cell countingvalues. The specific growth rate (p), defined as the increase in celldensity or dried biomass per unit time (Pirt 1975), was calculated andformulated as follows:μ=Ln(X1/X0)/(t1−t2)  (i)where X₀ and X₁ are cell density/dried biomass at the beginning (t₀) andend (t₁) of a selected time interval between inoculation and maximumcell density dried biomass, respectively. For the growth curve of eachsample, replicates were counted and the mean value used.

Microscopic study was done to evaluate the formation of palmella fromvegetative cells (two equal flagella). The density of palmella was foundto be 90% on the next sampling day of maximum specific growth in eachculture.

In the A culture group, more than 90% of palmella were found to beformed on days 16, 18 and 20 of culture in A1, A2, and A3 culture,respectively.

In the B culture group, a similar amount of percentage of palmella foundon 20, 22 and 24^(th) day of culture in ‘B1, B2, and B3, respectively.

Similarly, in the C culture group, it was observed on 20, 24 and 26^(th)day of culture in C1, C2 and C3, respectively (FIGS. 1, 2 and 3 ).

Cell abundance and Biomass production. Maximum specific growth rate(μ_(max) d⁻¹) is an informative way to ascertain microbial activitywhich can increase at exponential rates. Growth characteristicsdetermination under controlled conditions can play significantbiological information for mass culture of Haematococcus sp.

The μ_(max) d⁻¹ of Haematococcus sp. KAU-01 varied from 0.138 to 0.157(see bars in FIG. 1 ), 0.135 to 0.136 (see bars in FIG. 2 ) and 0.134 to0.147 d⁻¹ (see bars in FIG. 3 ) in culture groups ‘A’, ‘B’, and ‘C’,respectively. Among all cultures, the highest ρ_(max) d⁻¹ was in C1.

In the A culture group, the cell abundance varied from 15.45 to25.93×10⁵ cells mL⁻¹ with the highest in A3, followed by A2 and A1 (FIG.1 ).

In the B group, the cell abundance varied from 18.90 to 32.97×10⁵ cellsmL⁻¹, and the highest was in B3, followed by B2 and B1 (FIG. 2 ).

Similarly, in the C group, the cell abundance varied from 23.98 to40.92×10⁵ cells mL-1 with the highest in C3, followed by C2 and C1 (FIG.3 ). However, among all nine cultures, C3 had the highest cell abundanceof Haematococcus sp. KAU-01. Biomass production of Haematococcus sp.KAU-01 showed similar pattern as growth curve pattern of cell abundance.

Biomass production was found to be varied from 2.19 to 2.89 gL⁻¹ and thehighest was in A3, followed by A2 and A1 as shown by FIG. 4 .

In the B group, the highest biomass production was 3.28 gL⁻¹ in B3culture, followed by B2 (2.83 gL⁻¹) and B1 (2.57 gL⁻¹) as shown by FIG.5 .

Similarly, in C group, the biomass production varied from 2.89 to 4.26gL⁻¹ and the highest biomass production was found in C3 among C culturegroup (FIG. 6 ). However, the highest biomass production was found in C3culture among the three culture groups.

Increased biomass production of B1, B2 and B3 of B group was 0.35, 0.30and 0.90 gL⁻¹ higher than that the biomass production of A1, A2 and A3,respectively. The percentage calculation revealed that the biomassproduction of B1, B2 and B3 was 15.63, 11.68 and 37.90% more than thatthe biomass production of A1, A2 and A3, respectively.

Similarly, biomass production of C1, C2 and C3 group was 0.65, 0.69 and1.89 gL⁻¹ higher than that the biomass production of A1, A2 and A3,respectively. A percentage calculation revealed that the biomassproduction of C1, C2 and C3 was 29.03, 27.32 and 79.97% more than thatthe biomass production of A1, A2 and A3, respectively.

In medium replacement cultures, the increased biomass production was0.34 and 1.34 gL⁻¹ higher in B3 and C3 than that of the biomassproduction of A3, and it showed 11.71 and 45.79% high in B3 and C incomparison with A3; see Table 6 which shows the effects of culturing inthe presence of air (A), CO₂ (B), or combustion gas mixture (C).

TABLE 6 Comparison of biomass yields (g/L) for Haematococcus sp. KAU-01under conditions A (air), B (CO2) and C (combustion gas mixture).Increased biomass in Increased in ‘B’ Increased in ‘C’ Increased in ‘C’medium replacement compared to ‘A’ compared to ‘A’ compared to ‘B’cultures A3, B3 and C3 B-Cul gL⁻¹ % C-Cul gL⁻¹ % C-Cul gL⁻¹ % gL⁻¹ % B10.35 15.63 C1 0.65 29.03 C1 0.30 11.59 B3-A3 0.34 11.71 B2 0.30 11.68 C20.69 27.32 C2 0.40 14.00 C3-A3 1.34 45.79 B3 0.90 37.90 C3 1.89 79.97 C31.30 39.59

TABLE 7 Difference of biomass production gram per liter (gL⁻¹) andpercentage (%)during the culture of Haematococcus sp. KAU-01 underdifferent culture conditions A1, A2 or A3; B1, B2 or B3; or C1, C2 andC3. Difference of Biomass Difference of Biomass production (gL⁻¹)production in percentage (%) Different Cultures gL⁻¹ Percent calculation% (A2-A1) 0.31 {(A2-A1)/A1} × 100 13.77 (A3-A1) 0.66 {(A3-A1)/A1} × 10029.82 (A3-A2) 0.36 {(A3-A2)/A2} × 100 14.10 (B2-B1) 0.25 {(B2-B1)/B1} ×100 9.89 (B3-B1) 0.71 {(B3-B1)/A1} × 100 27.50 (B3-B2) 0.45 {(B3-B2)/B2}× 100 16.02 (C2-C1) 0.35 {(C2-C1)/C1} × 100 12.27 (C3-C1) 1.17{(C3-C1)/C1} × 100 59.49 (C3-C2) 1.35 {(C3-C2)/C1} × 100 42.06

Table 7 shows the difference of biomass production gram per liter (gL⁻¹)and percentage (%) within each of the A, B and C groups thus showing theeffects of different culture conditions 1, 2 or 3 on biomass production.Increased biomass production was observed in A2 and A3 which was 0.31and 0.66 gL⁻¹, and it was 13.77 and 29.82% when compared with thebiomass production of A1. In A3, the biomass production was 0.36 gL⁻¹and 14.10% higher than that of the biomass production of A2. Similarly,in B2 and B3 the biomass production was 0.71 and 0.45 gL⁻¹ more incomparison with the biomass production of B1, and it was 9.89 and 27.50%than that of B1. Biomass production of B3 was 0.45 gL⁻¹ and 16.02% thanthat of the biomass production of B2.

In the C culture group, the biomass production 1.17 and 1.35 gL⁻¹ washigher in C2 and C3, respectively, in comparison with the biomass of C1,and it was 12.27 and 59.49% when compared with the biomass production ofC1. Similarly, biomass production in C3 was 1.35 gL⁻¹ than that of thebiomass production of C2, and it was 42.06% higher when compared withthe biomass production of C2; see Table 7.

Effects of Replacement of Culture Liquids.

The effects of replacement of liquid in the culture to reduceconcentrations of cell debris, such as broken cells or cell walls, whichcan act as autoinhibitors of growth, were investigated. Soon afterreplacement, the culture medium, the discharged old culture medium wasfiltered following the same methods used to estimate cell biomass. Theconcentration of debris was 0.106, 0.110 and 0.120 gL⁻¹ in A3, B3 andC3, respectively, at the end of the study; FIG. 7 .

Cultures of Haematococcus sp. KAU-01 made using batch, fed batch, andculture medium replacement were grown with normal aeration, CO₂ andmixture gases supplying in certain interval along with normal aerationto maximize cell productivity and ultimately to obtain higher biomassproduction.

In all culture conditions Haematococcus sp. KAU-01 grew well butproduced different cell abundances and quantities of biomass. Theincreasing of cell abundance was arrested soon after stationary phasewhen above 90% free moving cells turned into cysts or palmella.

In batch cultures having only normal aeration and/or CO₂ and gasmixtures supplied along with normal aeration cultures, cysts wereobserved earlier than in fed batch cultures and in culture mediumreplacement cultures.

In batch culture with normal aeration (A1) and with supply of CO₂ (B1)and mixture gases (C1), the cyst formation observed on 16^(th) and20^(th) day of culture, respectively.

In fed batch culture with normal aeration (A2) and with supply of CO₂(B2) and mixture gases (C2), the cyst formation was observed on 22^(th)day of culture, respectively.

In replacement culture system, the cyst formation was observed on 20, 22and 24^(th) day of culture in A3, B3 and C3, respectively.

However, in replacement of culture system, the formation of palmella orcysts took longer time with normal aeration than that of the batch andfed batch cultures. Cyst formation took longest time in C3 culture,followed by B3 culture where mixture gases and CO₂ were supplied alongwith normal after replacement of culture medium. Here, the cystformation time was longer those reported by Kaewpintong et al. who foundformation of palmella from flagellated vegetative cell after 13^(th) dayof cultivation in M1, F1 and Hong Kong media at high-density cultivationof vegetative cells of H. pluvialis in airlift bioreactor; Kaewpintonget al., Photoautotrophic high-density cultivation of vegetative cells ofHaematococcus pluvialis in airlift bioreactor, BIORESOURCE TECHNOLOGY,January 2007, 98(2): 288-295.

Culture of H. pluvialis in different culture media and light intensitiesalso showed a stationary phase between 10 to 12 days which was muchlower than the inventors' culture systems; Imamoglu, E., et al. Effectof Different Culture Media and Light Intensities on Growth ofHaematococcus pluvialis. INTERNATIONAL JOURNAL OF NATURAL ANDENGINEERING SCIENCES, 2007, 3, 05-09. Medium replacement culturessupplied with CO₂ and gas mixtures showed almost double capacity to formcysts than that of cyst formation than in the culture study ofKaewpintong et al., supra and Imamoglu, E., et al., supra.

Cell growth generally occurs when vegetative cells are flagellated. Inpalmella (cysts) the cells convert to an inactive form and cease furthercell division. These results show that culture medium and conditionswere quite suitable and facilitated cell division for a long time, thusultimately increasing of cell abundance and biomass production.Moreover, it showed that replacement of culture medium with supplying ofCO₂ and mixture gases along with normal aeration provided a higherefficiency to facilitate more cell division by providing suitableconditions for long time than in batch and fed batch culture systems.

Cell abundance and Biomass production. The inventors sought to acquirebiological information, such as growth characteristics of Haematococcussp. KAU-01 to discover mass culture to construct a high-density massculture system. The μ_(max)d⁻¹ was significantly (p<0.05) higher in ‘C’culture group than that of the μ_(max)d⁻¹ of ‘A’ and ‘B’ culture group,except for ‘A1’ culture, where the maximum cell growth and stationaryphase were appeared about a week before than all other cultures. Theμ_(max)d⁻¹ in all cultures of this study was low in comparison with thestudy of Kaewpintong et al., supra who found μ_(max)d⁻¹ of 0.21 d⁻¹ inF1 culture medium on 8^(th) day of culture. Tjahjono et al. found themaximum specific growth rate of H. pluvialis was about 0.25 d⁻¹ usingbasal growth medium grown in mixotrophic condition with sodium acetateas a carbon source on 3^(rd) day of culture; Tjahjono et al.,Hyper-accumulation of astaxanthin in a green alga Haematococcuspluvialis at elevated temperatures, BIOTECHNOLOGY LETTERS. 1994,16:133-138.

The μ_(max)d⁻¹ can vary from culture to culture as well as initial cellabundance and occurrence of maximum cells abundance. However, the cellabundance was significantly higher (p<0.05) in all medium replacementcultures, followed by fed batch culture and batch culture. In batchculture with supplying of normal aeration, periodic supply of CO₂ andmixture gases along with normal aeration, the maximum cell abundance was15.54×10⁵, 18.90×10⁵ and 23.98×10⁵ cells mL⁻¹ in A1, B1 and C1 cultures,respectively (FIGS. 1, 2 & 3 ). Within batch culture, supply of mixturegases, CO₂ and normal aeration also showed significant (p<0.05)difference in cell abundance occurrence.

The cells abundance of batch cultures of normal aeration and supplyingmixture gases, CO₂ along with normal aeration showed almost two to threefolds higher than that of the results of obtained by Imamoglu et al.,supra who reported that the maximum cell abundance occurred 7.75×10⁵ and8.10×10⁵ cells mL⁻¹ in BG11 and RM culture media, respectively under thelight intensity of 50 μmol photons m⁻² s⁻¹ and in RM medium, therecorded cell abundance was 9.50×10⁵ cells mL⁻¹ when culture under 40μmol photons m⁻² s⁻¹.

In fed batch cultures, the maximum cell abundance was 19.95×10⁵,25.17×10⁵ and 31.50×10⁵ cells mL⁻¹ in A2, B2, and C2 cultures,respectively (FIGS. 1, 2 and 3 ). Within fed batch cultures, theoccurrence of cell abundance was also significantly difference,especially cell abundance in C2 was significantly (p<0.05) higher thanthat of B2, followed by A2 culture. Kaewpintong et al. studiedhigh-density cultivation of vegetative cells of H. pluvialis in airliftbioreactor with supplying CO₂ and attained the maximum cell abundance46×10⁴ and 79.5×10⁴ cells mL⁻¹, which is much lower than that of thecell abundance of the batch culture disclosed herein with periodicsupplying of CO₂ and mixture gases as well as fed batch culture withsupplying of CO₂ and mixture gases. The cell abundance was higher in allbatch cultures in comparison with the above mentioned previous study.

This is consistent with the AAHKAU medium creating favorablecircumstances for the higher growth of Haematococcus sp. KAU-01 in batchand fed batch culture with normal aeration or with periodic supply ofCO₂ and mixture gases along with normal aeration. Cultures supplied withmixture gases showed more cell abundance than all other cultures.Moreover, the remarkable maximum cell abundance occurrence was found ina 20% replacement culture medium. The cell abundance was 25.93×10⁵,32.97×10⁵ and 40.92×10⁵ cells mL⁻¹ in culture of A3, B3, and C3,respectively (FIGS. 1, 2 & 3 ). The cell abundance occurrence results inthe medium replacement culture was much higher than those reported byHata, N., et al., Production of astaxanthin by Haematococcus pluvialisin a sequential heterotrophic-photoautotrophic culture. JOURNAL OFAPPLIED PHYCOLOGY. 2001, 13, 395-402.

Biomass production. Biomass production was found to be high in all mediareplacement cultures. The 1^(st) highest was 4.26 gL⁻¹ in C3 culturewhere mixture gases was supplied along with normal aeration, and the 2ndhighest was in B3 culture where CO₂ gas was supply in same way to themedium replacement culture. This biomass production was higher incomparison with the biomass production of H. lacustris culture in NH₄Clenriched medium with supply of 2% CO₂ as bubbles, where the biomassproduction was 3.74 gL⁻¹.

Higher biomass production in CO₂ and mixture gases supplying culturesconfirmed that CO₂ and mixtures gases were converted to algal biomass byincreasing the components for photosynthesis.

It was also found that the biomass production in cultures supplied withmixture gases showed more biomass production than that of the biomassproduction by only supplying CO₂.

Mixture gases which are similar to flue gases (greenhouse gases) can beadded as a gas or in bicarbonate form as cultivated microalgae grow toofast to be able to take sufficient flue gases from the water. Compressedair can be blended and provided for algal photosynthesis. Therefore,high valued microalgae can be grown in high cell density for CO₂sequestration from flue gases, which ultimately reduces the emission offlue gases from fossil fuel-fired power plants.

These results demonstrate the remarkable growth of a commerciallyvaluable microalgae Haematococcus sp. KAU-01 in newly formulated AAHKAUculture medium with normal aeration as well as in cultures having aperiodic supply of CO₂ and/or mixture gases along with normal aeration.Superior results were obtained by replacement of culture medium toremove microalga autoinhibitors from ongoing cultures. This technologycan be used for profitable, large scale biomass production whilesimultaneously controlling undesirable emissions of flue gases from thefossil fuel power plants.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. All rangesprovided within the application are inclusive of the values of the upperand lower ends of the range unless specifically indicated otherwise.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include “A and B”, “A or B”, “A”, and “B”.

“About” means either within 10% of the stated value, or within 5% of thestated value, or in some cases within 2.5% or 1% of the stated value,or, “about” can mean rounded to the nearest significant digit.

The terms “naturally-occurring”, “native”, and “wild-type” refer to aform of Haematococcus sp. KAU—found in nature. For example, a naturallyoccurring or wild-type isolate of Haematococcus sp or a genetically orepigenetically unmodified parent strain to Haematococcus sp. KAU.

“Epigenetic modifications” are persistent and heritable changes made tothe DNA, which regulate how genes are expressed, but do not affect thenucleotide sequence itself. Epigenetic modifications include DNAmethylation, histone modification, and microRNA. A micro RNA(abbreviated miRNA) is a small non-coding RNA molecule (containing about22 nucleotides) found in plants, animals and some viruses, thatfunctions in RNA silencing and post-transcriptional regulation of geneexpression regulation.

“Exogenous nucleic acid molecule”, “transgene”, or “exogenous gene”refers to a nucleic acid molecule or gene that has been introduced(e.g., transformed) into a cell such as into Haematococcus sp. KAU. Atransformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. A descendent of a celltransformed with a nucleic acid molecule is also referred to as“transformed” if it has inherited the exogenous nucleic acid molecule.An “endogenous” nucleic acid molecule, gene or protein is a nativenucleic acid molecule, gene, or protein as it occurs in, or is naturallyproduced by, the host.

The term “recombinant protein” as used herein refers to a proteinproduced by genetic engineering regardless of whether the amino acidsequence varies from that of a wild-type protein or Haematococcus sp.KAU-01.

When applied to organisms, such as variants of Haematococcus sp. KAU-01,the term recombinant, engineered, or genetically engineered refers toorganisms that have been manipulated by introduction of a heterologousor exogenous recombinant nucleic acid sequence into the organism (e.g.,a non-native nucleic acid sequence), and includes gene knockouts,targeted mutations, gene replacement, and promoter replacement,deletion, disruption, or insertion, as well as introduction oftransgenes or synthetic genes or nucleic acid sequences into theorganism. That is, recombinant, engineered, or genetically engineeredrefers to organisms that have been altered by human intervention.Recombinant or genetically engineered organisms can also be organismsinto which constructs for reduced gene expression or gene “knockdown”have been introduced. Such constructs include, but are not limited to,RNAi, microRNA, shRNA, siRNA, antisense, and ribozyme constructs. Alsoincluded are organisms whose genomes have been altered by the activityof meganucleases, zinc finger nucleases, TALENs, or Cas/CRISPR systems.An exogenous or recombinant nucleic acid molecule can be integrated intothe recombinant/genetically engineered organism's genome or in otherinstances may not be integrated into the host genome. As used herein,“recombinant microorganism” or “recombinant host cell” includes progenyor derivatives of the recombinant microorganisms of the invention.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny or derivativesmay not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “heterologous” when used in reference to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme that is from a source orderived from a source other than Haematococcus sp. KAU-01 or that has anon-identical nucleic acid sequence or epigenetic form. In contrast a“homologous” polynucleotide, gene, nucleic acid, polypeptide, or enzymeis used herein to denote a polynucleotide, gene, nucleic acid,polypeptide, or enzyme that is derived from the host organism species.When referring to a gene regulatory sequence or to an auxiliary nucleicacid sequence used for maintaining or manipulating a gene sequence (e.g.a promoter, a 5′ untranslated region, 3′ untranslated region, poly Aaddition sequence, intron sequence, splice site, ribosome binding site,internal ribosome entry sequence, genome homology region, recombinationsite, etc.), “heterologous” means that the regulatory sequence orauxiliary sequence is not naturally associated with the gene with whichthe regulatory or auxiliary nucleic acid sequence is juxtaposed in aconstruct, genome, chromosome, or episome. Thus, a promoter operablylinked to a gene to which it is not operably linked to in its naturalstate (i.e. in the genome of a non-genetically engineered organism) isreferred to herein as a “heterologous promoter,” even though thepromoter may be derived from the same species (or, in some cases, thesame organism) as the gene to which it is linked.

As used herein, the term “protein” or “polypeptide” is intended toencompass a singular “polypeptide” as well as plural “polypeptides,” andrefers to a molecule composed of monomers (amino acids) linearly linkedby amide bonds (also known as peptide bonds). The term “polypeptide”refers to any chain or chains of two or more amino acids, and does notrefer to a specific length of the product. Thus, peptides, dipeptides,tripeptides, oligopeptides, “protein,” “amino acid chain,” or any otherterm used to refer to a chain or chains of two or more amino acids, areincluded within the definition of “polypeptide,” and the term“polypeptide” can be used instead of, or interchangeably with any ofthese terms. Polypeptides of Haematococcus sp. KAU-01 include thoseenzymes required for carotenoid production.

As used herein carotenoids include lutein, β-carotene, violaxanthin,neoxanthin, and zeaxanthin, chlorophyll a and b and other carotenoidsproduced by Haematococcus sp. KAU—or variants thereof.

As used herein, “expression” includes the expression of a gene at leastat the level of RNA production, and an “expression product” includes theresultant product, e.g., a polypeptide or functional RNA (e.g., aribosomal RNA, a tRNA, an antisense RNA, a micro RNA, a shRNA, aribozyme, etc.), of an expressed gene. The term “increased expression”includes an alteration in gene expression to facilitate increased mRNAproduction and/or increased polypeptide expression. “Increasedproduction” includes an increase in the amount of polypeptideexpression, in the level of the enzymatic activity of a polypeptide, ora combination of both, as compared to the native production or enzymaticactivity of the polypeptide. Thus, a modified Haematococcus sp. KAU-01may express less or more of a particular mRNA, protein or other valuableproduct such as a carotenoid like astaxanthin.

Some aspects of the present invention include the partial, substantial,or complete attenuation, deletion, silencing, inactivation, ordown-regulation of expression of particular polynucleotide sequences.The genes may be partially, substantially, or completely deleted,silenced, inactivated, or their expression may be down-regulated inorder to affect the activity performed by the polypeptide they encode,such as the activity of an enzyme. Genes can be partially,substantially, or completely deleted, silenced, inactivated, ordown-regulated by insertion of nucleic acid sequences that disrupt thefunction and/or expression of the gene (e.g., viral insertion,transposon mutagenesis, meganuclease engineering, homologousrecombination, or other methods known in the art). The terms“eliminate,” “elimination,” and “knockout” can be used interchangeablywith the terms “deletion,” “partial deletion,” “substantial deletion,”or “complete deletion.” In certain embodiments, a microorganism ofinterest may be engineered by site-directed homologous recombination ortargeted integration or mutation using a Cas/CRISPR system to knockout aparticular gene of interest. In still other embodiments, targetedinsertion into or mutation of a gene regulatory region using aCas/CRISPR system, RNAi, or antisense DNA (asDNA) constructs may be usedto partially, substantially, or completely silence, inactivate, ordown-regulate a particular gene of interest in Haematococcus sp. KAU-01.

The insertions, deletions, or other modifications of certain nucleicacid molecules or particular polynucleotide sequences in Haematococcussp. KAU-01 may be understood to encompass “genetic modification(s)” or“transformation(s)” such that the resulting strains of themicroorganisms or host cells may be understood to be “geneticallymodified”, “genetically engineered” or “transformed.”

As used herein, “enhancing the expression” includes an increase inexpression of a gene or nucleic acid molecule of interest or theactivity of an enzyme in a modified Haematococcus sp. KAU-01 comprisingone or more genetic modifications as compared to the expression oractivity in a control, unmodified Haematococcus sp. KAU-01 without suchgenetic modifications.

Reference to properties or compositions that are “substantially thesame” or “substantially identical” indicates minor and irrelevantdeviations that are not material to the characteristics consideredimportant in the context of the invention. In various embodiments thiscan mean the properties are within 10%, and preferably within 5%, within2.5% or within 1%, of the reference value. For example, AAHKAU mediumwith a variation of 1% in the content of one or more ingredients couldbe considered substantially the same as AAHKAU medium formulationdescribed herein.

The materials, methods, and examples are illustrative only and are notintended to be limiting. Other features and advantages of the inventionwill be apparent from the description and from the claims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A Haematococcus sp. that is Haematococcussp. KAU-01 deposited under Patent Deposit Number PTA-12772 or asubculture or an engineered variant thereof that has genomic orribosomal DNA at least 99% identical to that of Haematococcus sp.KAU-01.
 2. The Haematococcus sp. of claim 1 that is an engineeredvariant of Haematococcus sp. KAU-01 or a subculture thereof that has hadis genomic DNA epigenetically modified to differ from Haematococcus sp.KAU-01 deposited under Patent Deposit Number PTA-12772.
 3. TheHaematococcus sp. of claim 1 that is Haematococcus sp. KAU-01 depositedunder Patent Deposit Number PTA-12772 that has been transformed with anexogenous polynucleotide.
 4. A composition comprising Haematococcus sp.that is Haematococcus sp. KAU-01 deposited under Patent Deposit NumberPTA-12772 or subculture thereof in combination with an artificialculture medium that incorporates at least one material comprising aproduct obtained by combustion.
 5. The composition of claim 4, whereinthe artificial culture medium comprises: 375.00 mg/L NaNO₃, 75.00 mg/LKNO₃, 25.00 mg/L Ca(NO₃)₂, 55.00 mg/L Mg(NO₃)₂·6H₂O, 10.00 mg/L K₂SO₄,45.00 mg/L K₂HPO₄, 40.00 mg/L KH₂PO₄, 23.75 mg/L MgSO₄·7H₂O 1.755 mg/Lurea, 65 μl/L in HNO₃, 15 μl/L H₃PO₄; 3.50 mg/L FeCl₃·6H₂O, 1.00 mg/LH₃BO₃, 0.25 mg/L Co(NO₃)₂·6H₂O, 0.10 mg/L K₂Cr₂O₇, 0.10 mg/L CuSO₄·5H₂O,0.25 mg/L MnSO₄·H₂O, 0.75 mg/L ZnSO₄·6H₂O, 0.25 mg/L (NH₄)₆Mo₇O₂₄·4H₂O1.00 mg/L Na₂-EDTA and 1.00 μl/L in HCl; wherein one or more of saidconcentrations may vary by up to ±10%.
 6. The composition of claim 4,wherein the at least one material is taken from coal power plant fluegases.
 7. The composition of claim 4, wherein the at least one materialis obtained by combustion of coal and is obtained from a flue gasscrubber.
 8. The composition of claim 4, wherein the at least onematerial is taken from coal power plant flue gases with pressure into areserve tank and then supplied to the artificial medium comprising theHaematococcus sp.
 9. The composition of claim 4, wherein at least onematerial is incorporated into a gas, liquid or solid prior toincorporating it into the culture medium.
 10. The composition of claim4, wherein the at least one material is obtained by combustion ofnatural gas, petroleum, fuel oil, wood, or cellulose wastes.
 11. Thecomposition of claim 4, wherein the at least one material comprisescarbon dioxide.
 12. The composition of claim 4, wherein the at least onematerial comprises carbon monoxide.
 13. The composition of claim 4,wherein the at least one material comprises a nitrogen oxide.
 14. Thecomposition of claim 4, wherein the at least one material comprises asulfur oxide.
 15. The composition of claim 4, wherein the at least onematerial is a volatile organic compound.
 16. The composition of claim 4,wherein an amount of at least one heavy metal present in the at leastone material is reduced prior to incorporating it into the culturemedium.
 17. The composition of claim 4, wherein the Haematococcus sp.that has been passaged under conditions where more than 80% of the cellsdied and has subsequently become genetically or epigenetically stable.18. The composition of claim 4, wherein the subculture of Haematococcussp. has been mutated to reduce CHG DNA methylation compared to theunmutated strain.
 19. An engineered variant of Haematococcus sp. KAU-01deposited under patent deposit number PTA-127272; wherein said varianthas genomic DNA that is less than 100% identical to that ofHaematococcus sp. KAU-01 and wherein said variant has been produced bytransformation of Haematococcus sp. KAU-01 with an exogenous DNA, hasbeen produced by chemical or radiological treatment of Haematococcus sp.KAU-01, has been produced by having the genomic DNA of Haematococcus sp.KAU-01 epigenetically modified to differ from that of Haematococcus sp.KAU-01, wherein said variant has 18S and/or 26S rDNA that is 100%identical to that of Haematococcus sp. KAU-01.